DU/AM 


MOLOGT  LIBRAE 


THE   BOTANICAL  LIBRARY 

OF  THE 

UNIVERSITY  OF  CALIFORNIA. 

GIFT  OF 

MR.  AND    MRS.  T.   S.   BRANDEGEE. 
1906 


BOTANICAL 

MICRO-CHEMISTRY 

AN 

INTRODUCTION  TO  THE   STUDY  OF 
VEGETABLE  HISTOLOGY 

PREPARED  FOR  THE  USE  OF  STUDENTS 
BY 

V.  A.  POULSEN 


TRANSLATED  WITH  THE  ASSISTANCE  OF  THE  AUTHOR 
AND  CONSIDERABLY  ENLARGED 

BY 

WILLIAM  TRELEASE 

PROFESSOR  IN   THE   UNIVERSITY   OF  WISCONSIN 


Boston 

S.  E.  CASSINO  AND   COMPANY 
1884 


BIOLOGY 


Copyright, 
BY  S.  E.  CASSINO  &  CO. 

1883. 


ELECTROTYPED. 


BOSTON  STEREOTYPE  FOUNDRY, 
No.  4  PEARL  STREET. 


TO 


DOCENT  EUG.  WARMING,   Ph.D. 


JFrientr, 
PROFESSOR  LEOPOLD   KNY, 

THIS  BOOK   IS   DEDICATED  IN   THANKFULNESS 
BY  THE  AUTHOR. 


CONTENTS. 


PACK 

PREFACE  TO  AMERICAN  EDITION vii 

TRANSLATOR'S  PREFACE ix 

AUTHOR'S  PREFACE x 

INTRODUCTION xi 

LITERATURE  OF  BOTANICAL  MICRO-CHEMISTRY  xv 


MICRO-CHEMICAL  REAGENTS  AND  THEIR  APPLICATION  .       .  i 

APPENDIX. 

MOUNTING  MEDIA 65 

CEMENTS 71 


Part  QE, 

VEGETABLE  SUBSTANCES  AND  THE  MEANS  OF  RECOGNIZING 

THEM 75 


INDEX no 


SPECIAL    REAGENTS,    REACTIONS, 
AND   METHODS. 


PAGB 

Barcianu's  mucus  reaction     .    .  9 

Bar  feed's  glucose  test  ....  87 

Beale's  carmine 52 

De  Bary's  epiplasm  reaction  .  82 
Franchimont's  (Unverdor ben's) 

resin  test 94 

Grenacher's  alum-carmine  .  .  50 
Hanstein's  method  of  clearing 

tissues ii 

Hanstein's  aniline  violet  ...  59 

Hanstein's  metaplasm  reaction  .  82 

HohnePs  eerie  acid  test  .  .  .  18 

Hohnel's  xylofilin  reaction  .  .  77 

Maupas'  nucleus  staining ...  54 

Millon's  reagent 38 


PAGE 

Pringsheim's    hypochlorin    re- 
action        22 

Ranvier's  picro-carmine    ...  53 

Raspail's  protoplasm  test     .    .  44 

Russow's  potash  alcohol  ...  12 
Sachs'    reaction    for    starch  in 

chlorophyll  bodies    ....  7 

Schultze  maceration  fluid ...  34 

Stromeyer's  starch  reaction  .    .  4 
Treub's    method    of     clearing 

tissues 32 

Trommer's  sugar  test  .    .    .    .  35 
Unverdorben's  (Franchimont's) 

resin  test 94 

Wiesner's  lignin  test    ....  46 


PREFACE 

TO 

THE   AMERICAN    EDITION. 


THE  translation  of  the  Danish  original  into 
German,  Italian,  and  French,  and  now  into  Eng- 
lish, is  an  honor  not  dreamed  of  when  the  original 
edition  was  published. 

I  wish  to  cordially  thank  all  of  my  collaborators, 
especially  my  friends  Carl  Miiller  of  Berlin,  and 
Prof.  Aser  Poli  of  Melfi,  and  my  honorable  col- 
leagues Dr.  Lachmann  of  Lyon,  and  Prof.  Trelease 
of  Madison,  who  have  not  merely  enlarged  the 
book,  but  enriched  it  with  many  notes  and  para- 
graphs of  great  value. 

Once  more  I  wish  to  express  the  hope  that,  in 
the  new  shape  in  which  it  appears,  my  book  may 
be  of  use  to  the  students  of  vegetable  histology, 
for  whom  it  was  written. 

V.  A.  POULSEN. 
RQSENVAENGETS  HOVEDVEJ,  COPENHAGEN,  June  1883. 


TRANSLATOR'S   PREFACE. 


WHILE  studying  in  the  laboratory  of  Dr.  Far- 
low,  at  Harvard  University,  in  1881,  I  first  be- 
came acquainted  with  the  German  translation  of 
Poulsen's  little  treatise,  which  made  its  appear- 
ance in  this  country  about  that  time.  It  was 
proved  so  valuable  by  every-day  use  in  the  labora- 
tory, both  there  and  with  my  classes  in  the  Uni- 
versity of  Wisconsin,  that  I  began  a  translation  of 
the  most  frequently  used  parts,  having  in  mind  at 
first  only  the  convenience  of  my  own  students. 
But  several  requests  having  been  made  that  I 
should  complete  the  translation  and  publish  it,  I 
decided  to  do  so,  having  received  an  offer  of  addi- 
tional notes  from  the  author.  Various  causes,  how- 
ever, have  delayed  my  work,  so  that  it  is  but  now, 
above  a  year  since  it  was  begun,  that  it  is  completed. 

It  should  be  said  that  the  translation  is  in  no 
sense  a  literal  one.  The  obvious  meaning  of  the 
author  has  been  given,  but  with  no  effort  to  pre- 
serve the  idiom  or  vocabulary,  and  the  usual  Eng- 
lish terminology  has  commonly  been  employed. 

In  presenting  my  translation  to  American  teach- 
ers of  vegetable  histology,  I  feel  confident  that  it 
will  meet  the  every-day  wants  of  the  laboratory. 

WM.  TRELEASE. 

MADISON,  Wis.,  April  12,  1883. 


AUTHOR'S   PREFACE. 


THIS  little  attempt  at  a  comprehensive  pre- 
sentation of  the  more  important  micro-chemical 
reagents  and  methods  is  the  first  which  has 
appeared  in  the  Danish  language.  It  has  been 
undertaken  on  the  recommendation  of  Dr.  Eug. 
Warming,  who  has  often  felt  the  need  of  a  com- 
pact guide  to  micro-chemistry  in  his  work  with 
students  of  vegetable  anatomy  at  the  Botanic 
Garden.  To  him  I  am  indebted  for  many  valuable 
suggestions,  which  I  find  it  a  pleasant  duty  to 
acknowledge  here. 

I  am  also  indebted  to  Mr.  R.  Pedersen,  Docent 
in  Vegetable  Physiology  at  the  Copenhagen  Uni- 
versity, for  many  criticisms. 

Should  those  inexperienced  in  microscopy  draw 
even  a  little  useful  information  from  this  treatise 
I  shall  have  accomplished  what  was  aimed  at. 

V.  A.  POULSEN. 

ROSENVAENGET,  October  1880. 


INTRODUCTION. 


RECENTLY  microscopic  technology  has  risen  to 
an  importance  undreamed  of  in  its  early  days. 
The  perfection  of  the  microscope,  by  whose  help 
so  many  beautiful  and  important  discoveries  are 
made,  has  given  us  such  an  insight  into  the  nat- 
ural history  of  the  cell  as  to  stimulate  the  study 
of  elementary  organs,  so  far  as  possible,  with 
all  available  aids.  The  spectroscope,  the  polar- 
iscope,  and  the  induction  coil,  placed  in  our  hands 
by  Bunsen,  Huygens,  and  Faraday,  are  applicable 
and  must  augment  the  value  of  the  instrument 
first  given  us  by  Hans  and  Zacharias  Janssen ; 
and  even  photography  has  of  late  been  employed. 
Physics  has  thus  striven  to  bring  this  instrument 
to  as  great  a  degree  of  perfection  as  possible ; 
it  remains  for  chemistry  to  find  means  of  recog- 
nizing and  rightly  understanding  the  composition 
of  the  objects  we  investigate.  In  other  words,  if 
we  employ  a  thorough  system  of  chemical  analysis 
with  the  optical  apparatus  we  shall  be  able  to 
answer  all  questions  lying  within  the  range  of  pos- 
sibility. It  is  this  analysis,  applied  to  objects 

xi 


Xll  INTRODUCTION. 

under  the  microscope,  that  we  designate  by  the 
word  micro-chemistry. 

I  have  endeavored  to  successively  make  the 
reader  acquainted  with  the  most  valuable  reagents 
used  in  micro-chemistry,  i.  e.  with  those  sub- 
stances whose  action  on  the  bodies  to  be  studied 
allows  their  chemical  composition  and  nature, 
and  sometimes  their  physical  structure,  to  be 
recognized.  In  the  first  section  I  have  consid- 
ered the  chemicals  used  in  the  laboratory ;  in  the 
second,  the  vegetable  substances  to  be  tested  for 
and  the  reactions  by  which  they  are  known. 

The  correctness  of  the  statements  which  follow 
rests  in  part  on  the  long  experience  of  my  re- 
spected teacher,  Dr.  Warming,  and  in  part  on 
my  own.  I  have  also  been  able  to  profit  by  the 
practice  and  teaching  of  Professor  Hanstein,  of 
Bonn,  made  known  to  me  by  Dr.  Warming. 
Finally,  the  scattered  experiments  and  methods, 
recorded  in  a  large  and  scattered  literature,  have 
been  used  so  far  as  possible, — far  be  it  from  me, 
however,  to  suppose  that  I  have  exhausted  the 
literature ;  nor  have  I  tried  to  take  up  all  of  the 
chemicals  that  have  been  used,  my  endeavor  being 
to  collect  only  such  as  are  most  useful. 

At  the  close  of  the  first  section  I  have  intro- 
duced a  short  chapter  on  media  for  the  preserva- 
tion of  permanent  preparations ;  to  which  are 
added  a  few  words  on  the  cements  used  in 
mounting. 


INTRODUCTION.  Xlll 

Pleasant  as  it  would  have  been  to  add  a  histori- 
cal outline  of  the  development  of  micro-chemistry, 
I  have  thought  best  not  to  do  so,  partly  that  the 
book  might  be  kept  within  proper  limits  as  to  size, 
partly  because  that  treatment  of  the  subject 
would  more  properly  find  place  in  a  theoretical 
and  comprehensive  text-book  than  in  a  compact 
guide  for  purely  practical  use. 


LIST  OF  THE  MORE  IMPORTANT  PUBLICA- 
TIONS WHICH  HAVE  BEEN  USED  IN  THE 
PREPARATION  OF  THIS  BOOK. 


Almquist :  Metoder  attodla  och  farga  Bakterier.  —  Hygiea,  1883,  Stockholm. 
Bachmann :  Leitfaden  zur  Anfertigung  mikroskopischer  Dauerpraparate, 

Miinchen,  1879. 
Bary,  De :  Morphologic  und  Physiologic  der  Pilze,  Flechten  und  Myxomy- 

ceten.  —  Hofmeister's  Handbuch,  Bd.  II.,  Abth.  i ;  Vergleichende  Anato- 
mic.—  Hofmeister's  Handbuch,  Bd.  III.,  1877. 
Behrens:   Die  Nectarien  der  Bliithen. —  Flora,  1879;  Hilfsbuch  f .  mikro- 

skop.  Untersuchungen,  1883. 
Berg:  Zur  Kenntniss  des  in  der  Cetraria  islandica vorkommenden  Lichenins 

und  jodblauenden  Stoffes,  Dorpat,  1872. 

Bonnier:   Les  Nectaires.  —  Ann.  Sci.  Nat,  Bot.,  1879,  6  Se"r.,  Tome  VIII. 
Burgerstein:   Sitzungsber.  wien.  Akad.,  1874,  Bd.  LXX.,  p.  338. 
Cario:  Tristicha  hypnoides.  —  Bot.  Zeitung,  1881,  p.  31. 
Dippel:  Das  Mikroskop,  I.  and  II.,  1869;  Die  neuere  Theorie  iiber  die 

feinere  Struktur  der  Zellhiille,  1878. 
Eriksson :  Om  Meristemet  i  dicotyla  vaxters  rotter.  —  Lunds  Universitets 

arsskrift,  1877,  XIII.,  p.  10. 
Errera:  L'epiplasme  des  Ascomycetes  et  le  glycogene  des  Vegetaux,  Brux- 

elles,   1882 ;  Glycogene  des   Mucorine'es.  —  Bull.  Acad.  de   Belgique, 

1882. 
Flemming:  Ueb.  das  Hermannsche   Kernfarbungsverfahren.  —  Archiv.  f. 

mikr.  Anat,  1881,  XIX. 

Fritsche:  Ueber  den  Pollen.  — Mem.  Acad.  des  Sci.  de  St.  Petersbourg,  1837. 
Hanstein :   Die  Milchsaftgefasse  und  verwandte  Organe  der  Rinde,  1864; 

Die  Scheitelzellgruppe  im  Vegetationspunkt  der  Phanerogamen,  1868 ; 

Organe  der  Harz-und  Schleimabsonderung  in  den  Laubknospen.  —  Bot. 

Zeitung,  1868. 
Hartig :   Bot.  Zeitung,  1856,  p.  262  ;   Entwickelungsgeschichte  des  Pflan- 

zenkeimes,  Leipzig,  1858;  Der  Fiillkern,  etc. — Karsten's  bot.  Unter- 
suchungen, 1867, 1. 


XVI  LIST    OF    BOOKS    USED. 

Hegelmaier:    Bau  und    Entwickelung  einiger  Cuticulargebilde.  —  Jahrb. 

wiss.   Bot.,  IX.;    Vergl.   Untersuch.    iiber    Entwick.    dikot.    Keime, 

1878,  p.  ii. 
Higley :  Sur  1'acide  phosphorique  et  les  phosphates.  —  Bot.  Centralblatt, 

1881,  No.  27. 
Hofmeister:     Die    Pflanzenzelle.— Handbuch    der   physiol.   Bot.,   1867, 

Bd.  I. ;  Handbuch,  Bd.  II.  and  III.  —  See  De  Bary. 
Hohnel,  Von :  Ueber  Kork  und  verkorkte  Gewebe  iiberhaupt.  —  Sitzungs- 

ber.  wien.  Akad.,  1877,  Bd.  LXXVI.,  Abth.  i. 
Johow:  Zellkerne  der  hoheren  Monocotylen.,  Diss.,  Bonn,  1880. 
Kabsch:    Chemische   Beschaffenheit   der  Pflanzengewebe. — Jahrb.  wiss. 

Bot.,  1861,  III.,  p.  357. 

Kaiser:   Zeitschrift  fiir  Mikroskopie,  1877-78,  Bd.  I. 
Karsten  :  Gesammelte  Beitrage  zur  Anatomie  und  Physiologic  der  Pflan- 

zen,  1865, 1.,  p.  253. 
Koch :  Verfahren  zum  Conserviren  und  Photographiren  der  Bacterien.  — 

Cohn's  Beitrage  zur  Biol.  der  Pflanzen,  II.,  p.  399. 
Maschke:  Pigmentlosung  als   Reagenz  bei  mikroskopish-physiol.    Unter- 

suchungen.  —  Bot.  Zeitung,  1859,  p.  51. 
Meyen :  Anatom.-physiol.  Untersuchungen  iiber  den  Inhalt  der  Pflanzen- 

zellen,  Berlin,  1828. 
Mohl,  Von:  Vermischte  Schriften,  bot.  Inhalts,  Tubingen,  1845 ;  Aufbe- 

wahrung  micr.  Praparate. —  Bot.  Zeitung,  1857. 
Miiller,  N.  J.  C. :   Untersuchungen  iiber  d.  Vertheilung  der  Harz  etc.  im 

Pflanzenkorper.  —  Jahrb.  wiss.  Bot.,  V.,  p.  397  ;  Handbuch  der  Botanik, 

I. :   Allgem.  Botanik.  Erster  Theil :   Anatomie  und  Physiol.  der  Ge- 

wachse,  Heidelberg,  1880. 
Nageli :   Die  Starkekorner,  ZUrich,  1858.  . 
NageJi  and  Schwendener:   Das  Mikroskop,  1877,  2d  ed. 
Planeth:    Mikrochemische  Analyse  der  vegetabilischen    Zelle.  —  Promo- 

tionsschrift  an  der  Univers.  Rostock,  1873. 
Pfeffer:  Die  Proteinkorner.  —  Jahrb.  wiss.  Bot.,  1872,  VIII. 
Pfitzer:  Die  Bacillariaceen.  —  Hanstein's  bot.  Abhandl.,  1871, 1.,  Heft  2. 
Pringsheim:    Untersuchungen    iiber  das  Chlorophyll,  IV.,  Das    Hypo- 

chlorin. —  Monatsber.  berl.  Akad.,  Nov.  1879. 
Radlkofer:  Krystalle  Protemartige  Korper,  1859. 
Ranvier:  Trait6  technique  d'  histologie,  Paris,  1877, 
Robin  :   Trait6  du  microscope,  Paris,  1877. 
Russow :  Vergl.  Unters.  uber  Leitbiindel-Kryptogamen.  —  Mem.  Acad.  des 

Sci.  de  St.  Petersbourg,  1872,  7  Se"r.,  Tome  XIX.,  No.  i ;  ZUF  Kennt- 

niss  dss   Holzes,  insonderheit  des  Coniferenholzes.  —  Bot.  Centralbl., 

1883,  No.  i. 


LIST   OF   BOOKS   USED.  XV11 

Sachs:  Keimung  der  Graser;  Keim  der  Dattel. — Bot  Zeitung,  1862; 
Ueber  die  Stoffe,  welche  das  Material  zum  Wachsthum  der  Zellhaut 
liefern.  —  Jahrb.  fur  wiss.  Bot.,  1863,  III. ;  Lehrbuch  der  Botanik,  4th 
ed.,  1874 ;  Ueber  einige  -»eue  mikroskopisch-chemische  Reaktionsmeth- 
oden.  —  Sitzb.  wien.  Akad.,  1859  ;  Keimung  der  Schminkbohne.  —  Ibid. 

Sachsse :  Chemie  und  Physiol.  der  Farbstoffe,  Kohlenhydrate  und  Prote'm- 
substanzen,  Leipzig,  1877. 

Sanio-  Various  papers.  —  Bot.  Zeitung,  1860,  1863 ;  Anatomie  der  Kiefer. 

—  Jahrb.  fur  wiss.  Bot.,  1873-74,  IX. 

Schacht:  Die  Pflanzenzelle,  1852;  Das  Mikroskop,  1855;  Anatomie  und 
Phys.  der  Gewachse,  1856,  I. ;  Ueber  den  Bau  einiger  Pollenkorner. — 
Jahrb.  fur  wiss.  Bot.,  1860,  II.,  p.  109. 

Schimper,  A. :  Untersuchungen  iiber  Protein-krystalloide  der  Pflanzen, 
Strassburg,  1879. 

Schmitz :  Becbachtungen  iiber  die  vielkernigen  Zellen  der  Siphonocladia- 
ceen,  Halle,  1879. 

Strasburger:  Befruchtung  und  Zelltheilung,  1878;  Studien  iiber  Proto- 
plasma,  1876;  Zellbildung  und  Zelltheilung,  Jena,  1875;  3d  ed.,  1880. 

Tangl :  Das  Proto plasma  der  Erbse.  —  Sitzungsber.  der  wien.  Akad.,  1877- 
78,  Bd.  XXVI.  and  XXVIII. ;  Communication  zwischen  der  Zellen  des 
Endosperms.  —  Jahrb.  wiss.  Bot.,  1880,  XII.;  Beitrage  z.  Mikrochemie 
d  Pflanzenzellen.  —  Sitzb.  wien.  Akad.,  1876,  Abth.  i. 

Treub :  Me"risteme  primitif  de  la  racine  dans  les  Monocotyledones,  Leide, 
1876 ;  Sur  le  r61e  du  noyau  dans  la  division  des  cellules,  1878  ;  Sur  des 
cellules  vegetales  a  plusieurs  noyaux.  —  Archives  N6erlandaises,  T.  XV. 

—  Separate,  p.  15. 

Vogl:  Anatomie  und  Histologie  der  unterirdischen  Theile  von  Convol- 
vulus arvensis.  —  Sitzungsber.  wien.  Akad.,  1863,  Bd.  XIII.,  p.  257 ;  Bau 
des  Holzes  von  Ferreira  spectabilis. — Jahrb.  fiir  wiss.  Bot.,  1873- 
74,  IX. 

Vries,  De :  Keimungsgeschichte  des  rothen  Klees.  —  Landwirthschaftliche 
Jahrbiicher,  1877,  Bd.  VI. 

Weiss:  Die  Pflanzenhaare.  —  Karsten's  bot.  Untersuchungen,  1867,  I. ; 
Allgemeine  Botanik,  1878,  Bd.  I. 

Wiesner:  Technische  Mikroskopie,  Wien,  1867;  Anatomisches  und 
Histochemisches  iiber  das  Zuckerrohr.  —  Karsten's  bot.  Untersuch., 
1867,  I. ;  Das  Verhalten  des  Phloroglucins  und  eniger  verwandter 
Korper  zur  Zellmembran.  —  Sitzungsber.  wien.  Akad,  1878,  Abth.  i. 

Wigand:  Intercellularsubstanz  und  Cuticula,  1850. 


XV111  LIST   OF   BOOKS   USED. 


LITERATURE     OF    THE     COLORING    MATTERS 
FOUND     IN    PLANTS. 

(EXCLUSIVE  OF  PAPERS  ON  SPECTROSCOPIC  AND  OTHER 
OPTICAL  TESTS.) 

Askenasy :  Bot.  Zeitung,  1867,  p.  227 ;  1869,  p.  785. 

Brown,  R. :  Manual  of  Botany,  p.  589. 

Cohn :  Bot.  Zeitung,  1867,  p.  38. 

Fremy :  Annales  des  Sci.  Nat.,  Bot,  1860,  T.  XIII. 

Hildebrandt :  Die  Farben  der  Bluthen.  —  Jahrb.  fiir  wiss.  Bot.,  III. 

Kiitzing  :   Phycologia  generalis,  p.  17,  et  seq, 

Millardet:  Comptes  rend.,  1869.   (Cf.  Bot.  Zeitung,  1869,  p.  332.) 

Miiller,  N.  J.  C.:  Handb.  d.  Bot.,  Bd.  I.,  Allgem.   Bot.,  Theil   i,  1880, 

p.  562. 

Nageli  and  Schwendener:   Das  Mikroskop,  1877,  p.  528. 
Prantl :   Bot.  Zeitung,  1871,  p.  619. 

Pringsheim:  Monatsber.  berlin.  Akad.,  October  1874;  December  1875. 
Rosanoff :  Mem.  de  la  Soc.  de  Cherbourg,  1867,  XIII. ;  Bot.  Zeitung,  1866, 

p.  182. 
Sachsse :  Die  Farbstoff e,  Kohlenhydrate  und  Prote'insubstanzen,  Leipzig, 

1877;  Die  Chromatophoren  der  Algen,  Bonn,  1882. 
Timirjaseff :   Das  Chlorophyll,  1871. 
Trecul :  Annales  des  Sci.  Nat.,  Bot.,  1858,  4  Ser.,  T.  X. 
Weiss :   Allgemeine  Bot.,  1878,  L,  p.  106-138. 
Wiesner:   Bot.  Zeitung,  1862;  Flora,  1874. 

Besides  the  works  cited  here,  others,  which  have  been  used  occasionally, 
are  mentioned  in  various  places  in  the  text. 


PART  I 
MICRO-CHEMICAL     REAGENTS 

AND 

THEIR  APPLICATION. 


MICRO-CHEMICAL   REAGENTS. 


IODINE. 

THIS  is  one  of  the  most  valuable  and  indis- 
pensable substances  used  in  microscopic  botany. 
It  is  employed  in  solution,  either  in  water,1  alcohol, 
or  a  solution  of  potassic  iodide  in  glycerine,  chlor- 
iodide  of  zinc,  or  water.  The  degree  of  concentra- 
tion of  the  solution  depends  upon  the  particular 
case  in  which  it  is  to  be  used,  the  effects  of  much- 
diluted  solutions  being  often  very  evident.  For 
the  recognition  of  starch,  iodine  is  a  very  con- 
venient reagent,  and  the  only  certain  one  known. 
A  preparation  which  contains  the  minimum  of  free 
iodine  suffices  to  reveal  this  substance,  though  only 
in  case  the  starch  contains  water.  With  an  aque- 

1  Nageli:  Das  Mikroskop,  1877,  p.  473.  Dippel:  Das  Mikroskop, 
1872,  p.  273.  Sachs :  Jahrbiicher  fiir  wiss.  Bot.,  1863,  III.  Mohl :  Ver- 
mischte  Schriften,  1845,  p.  335.  Hofmeister:  Handbuch,  I.,  Pflanzenzelle, 
1867,  pp.  252,  387.  Weiss:  Allgemeine  Botanik,  1878,  I.,  pp.  18,  77,  159. 
Schleidcn  :  Wicgmann's  Archiv.,  1838,  pp.  39,  59.  Raspail .  Developpement 
et  Analyse  microscopique  de  la  Fecule.  —  Ann.  Sci  Nat ,  i  Ser.,  T.  VI., 
p.  388.  Meyen :  Untersuchungen  liber  den  Inhalt  der  Pflanzenzellen,  Ber- 
lin, 1828,  p.  21.  Sachs:  Lehrbuch  der  Botanik,  1874,  PP  34>  4°>  5^. 

3 


4  MICRO-CHEMICAL   REAGENTS. 

ous  solution,  this  condition  is  fulfilled  ;  but  in  case 
a  solution  in  absolute  alcohol  is  used,  water  must 
be  added  to  the  preparation. 

The  well-known  reaction,  discovered  by  Stro- 
meyer,  is  as  follows  : x  According  to  the  strength 
of  the  solution  and  the  duration  of  its  action,  the 
starch  grains  assume  a  more  or  less  deep-blue 
color,  which  vanishes  when  the  preparation  is 
warmed,  but  reappears  when  it  again  becomes 
cool,  and  is  entirely  destroyed  by  the  action  of 
hyposulphite  of  sodium.  Dry  starch  slowly  turns 
brown  when  treated  with  a  solution  of  iodine  in 
chloroform  or  absolute  alcohol,  which  renders  it  a 
good  test  for  the  presence  of  water  in  alcohol. 
According  to  Nageli,  the  starch  grains  are  com- 
posed of  granulose  diffused  through  a  skeleton  of 
starch-cellulose.  The  former,  only,  is  turned  blue 
by  iodine,  for  after  its  removal  (by  digestion  with 
saliva  at  45-55°  C.,  or  the  action  of  pepsin,  dias- 
tase, organic  acids,  or  the  prolonged  action  of  dilute 
sulphuric  or  hydrochloric  acid)  the  grains  turn  yel- 
low or  brown. 

Most  cellulose  membranes  color  yellow  or 
brownish  with  iodine,  especially  when  a  freshly- 
made  solution  is  employed.  An  exception,  how- 
ever, is  afforded  by  the  paraphyses  and  asci,  —  the 
hymenium  of  lichens,  which  often  turn  blue,  like 
starch.  If,  however,  the  cell-wall  has  been  first 

1  Nageli:  Die  Starkekorner,  Ziirich,  1858.  Wiesner:  Technische 
Mikroskopie,  1857,  p.  73. 


IODINE.  5 

treated  with  strong  sulphuric  or  phosphoric  acid,  or 
with  an  aqueous  solution  of  zinc  chloride,  it  colors 
blue  when  iodine  is  added,  these  reagents  convert- 
ing the  cellulose  into  amyloid, I  a  carbo-hydrate 
related  to  starch.  Hydriodic  acid  also  acts  in  the 
same  way,  and  hence  old  solutions  of  iodine  some- 
times color  the  cell-wall  blue,  when  this  acid  has 
formed  through  the  action  of  the  water  or  alcohol 
of  the  solution.  Cellulose  membranes  that  have 
been  allowed  to  dry  after  treatment  with  a  solu- 
tion of  iodine,  and  are  again  moistened  with  water, 
often  turn  blue,  the  organic  matter  having  in- 
duced the  formation  of  hydriodic  acid.  The  epi- 
dermal cell-walls  of  many  seeds  and  pericarps  that 
swell  into  mucilaginous  masses  when  moistened,  as- 
sume a  blue  color  with  iodine-water  only  after  the 
swelling  has  reached  a  certain  stage.  Not  unfre- 
quently  a  membrane  lying  in  an  iodine  solution 
passes  through  several  shades  of  color  in  the  course 
of  a  longer  or  shorter  time.  Most  reagents  that 
cause  cellulose  to  turn  blue  when  used  in  combina- 
tion with  iodine,  also  cause  it  to  swell,  approxi- 
mately, in  the  following  ascending  order :  Potassic 
iodide,  iodide  of  zinc,  nitric  acid,  phosphoric  acid, 
potassic  hydrate,  hydriodic  acid,  and  sulphuric 
acid. 

We  have  thus  an  excellent  test  for  pure  cellu- 
lose in  iodine  and  any  amyloid-producing  reagent. 

1  Not  to  be  confounded  with  the  substance  known  to  animal  histolo- 
gists  by  the  same  name. 


6  MICRO-CHEMICAL   REAGENTS. 

If,  however,  a  membrane  does  not  color  blue  when 
treated  with  sulphuric  acid  and  iodine,  it  is  per- 
meated by  other  substances,  or  is  to  be  regarded 
as  changed  chemically ;  this  is  true  of  wood-cells, 
vessels,  cork,  and,  usually,  the  root-cap.  The 
substances  (lignin,  suberin)  taken  up  by  the  walls 
in  these  cases  must  first  be  removed  by  the  alter- 
nate use  of  acids  and  alkalies,  and  finally  by 
washing  in  alcohol,  ether,  or  chloroform,  before 
the  cellulose  reaction  will  appear ;  but,  as  yet,  with 
the  single  exception  of  the  silicified  frustules  of 
diatoms,  no  cell-wall  has  been  found  that  is  not 
shown  by  this  treatment  to  contain  cellulose. 

In  the  study  of  lichens,  chemical  reagents  play 
an  important  part. '  The  preparation  usually  em- 
ployed, consists  of  5  eg.  iodine,  20  eg.  potassic 
iodide,  and  15  g.  distilled  water.  In  thin  sec- 
tions, the  hymenium  is  usually  colored  blue  by  this 
solution,  the  medullary  layer,  occasionally,  and  the 
gonidial  layer,  less  commonly.  A  wine  color  may 
also  appear  under  this  treatment,  which  shows 
that  these  cell-walls  have  a  composition  different 
from  those  of  other  plants. 

All  living  protoplasm2  is  killed  by  iodine,  which 
is  then  rapidly  imbibed  and  stains  the  protoplasm 

1  Deichmann-Branth  and  Rostrup :  Lichenes  Daniae,  p.  17.     De  Bary 
Morphol.  und  Physiol.   der    Pilze,  Flechten    und    Myxomyceten.  —  Hof- 
meister's  Handbuch,  II.,  1866,  p.  281. 

2  Sachs:    Lehrbuch,    1874.     Weiss:    Allgem.  Bot.,  1878,  I.     Tangl: 
Protoplasma    der    Erbse.  —  Sitzungsber.       wiener    Akad.,    1877-8,    Bd. 
LXXVI.-LXXVIII. 


IODINE.  7 

brown.  This  is  'especially  true  of  the  nucleus  and 
chlorophyll  bodies,  as  well  as  of  the  nitrogenous 
fundamental  part  of  the  protein  grains,  for  the 
detection  of  which  a  rather  concentrated  solution 
should  be ,  used.  Since  a  very  small  quantity  of 
iodine  is  quickly  fatal  to  protoplasm,  besides  col- 
oring it,  this  reagent  is  especially  useful  in  study- 
ing bacteria  and  other  ciliated  micro-organisms. 
In  the  study  of  protein  grains  the  glycerine  solu- 
tion is  best,  because  of  its  clearing  action. 

The  starch I  which  occurs  in  chlorophyll  bodies  is 
easily  detected  by  treating  very  thin  sections  with 
alcohol,  or  potassic  hydrate  and  acetic  acid,  and 
afterwards  adding  a  solution  of  iodine  in  water 
which  contains  potassic  iodide,  when  the  swelling 
starch  grains  assume  the  characteristic  blue  color. 

The  so-called  crystalloids  consist  of  protein  sub- 
stances, and  consequently  turn  yellow  when  treated 
with  iodine.  This  is  also  the  case  with  inuline, 
where,  however,  the  color  does  not  depend  upon  a 
real  imbibition  of  the  iodine,  but  only  upon  a  con- 
densation of  the  brown  fluid  in  the  fine  fissures  of 
the  sphaero-crystals. 

All  objects  colored  with  iodine  fade  in  the 
course  of  time ;  and  iodine  solutions  destined  for 
use  should  be  kept  in  the  dark,  to  prevent  the 
formation  of  hydriodic  acid,  which  is  greatly  pro- 
moted by  the  action  of  light.  When  sections  or 

1  Sachs:  JahrbUcher  fiir  wiss.  Bot,  1863,  III.,  p.  200;  Flora,  1862,  p. 
166.  Weiss:  Allgem.  Bot.,  1878, 1.,  p.  in. 


8  MICRO-CHEMICAL   REAGENTS. 

other  preparations  are  removed  from  water  into  a 
concentrated  alcoholic  tincture  of  iodine,  small, 
black,  rhombic  crystals  of  iodine  often  make  their 
appearance. 


CHLOR-IODIDE  OF  ZINC. 

In  the  preceding  section  mention  has  been  made 
of  a  solution  of  iodine  in  chloride  of  zinc.1  We 
may  now  consider  this  a  little  more  fully. 

This  preparation  is  made  by  dissolving  zinc  in 
pure  hydrochloric  acid,  evaporating  the  solution  to 
the  density  of  sulphuric  acid,  in  contact  with 
metallic  zinc,  and  adding  as  much  potassic  iodide 
as  the  solution  will  take  up.  Finally,  it  is  saturated 
with  metallic  iodine.2  The  color  of  the  reagent 
should  be  reddish-brown ;  it  should  have  the  odor 
of  iodine,  and  small  crystals  of  pure  iodine  should 
precipitate  with  time.  As  a  precaution  against 
the  formation  of  hydriodic  acid,  it  should  be  kept 
in  the  dark,  although  this  is  less  important  than 
with  the  other  iodine  preparations. 

Chlor-iodide  of  zinc  is  especially  useful  for  the 
detection  of  pure  cellulose,  since  the  zinc  chloride 

1  Nageli:  Verhalten  der  Zellhaut  zum    Jod.  —  Sitzungsber.  d  bayr. 
Akad.  der  Wiss.,  1863,  p.  383;  Das  Mikroskop,  1877,  p.  474.    Mohl:  Blaue 
Farbung  der  Vegetab.  Zellmembran  durch  Jod.  —  Flora,  1840.   See  also  the 
works  cited  above. 

2  The  directions  of   Gronland,  Cornu  and    Rivet  (Des  Preparations 
Microscopiques,  Paris,  1872,  p.  75),  are  incorrect,  since  the  most  important 
element  —  the  iodine  —  is  not  present. 


POTASSIC  HYDRATE.  9 

converts  this  substance  into  amyloid,  which  is 
then  colored  blue  or  violet  by  the  free  iodine. 
Cell-walls  that  have  suffered  degeneration  of  the 
cellulose  are  not  colored  blue.  Wood-cells,  ves- 
sels, cork-cells,  and  the  cells  of  the  root-cap,  as 
well  as  the  cuticularized  layer  of  the  epidermis, 
the  extine  of  pollen  grains  and  spores,  in  fine,  all 
lignified  or  corky  membranes,  are  colored  yellow ; 
the  true  cuticle,  however,  is  uncolored.  Starch 
colors  blue,  but  the  grains  rapidly  swell  up  and 
undergo  disorganization.  The  walls  of  fungus 
hyphae,  composed  of  the  so-called  fungus-cellulose, 
remain  uncolored  to  a  noticeable  degree.1  [They 
are  also  usually  uncolored  by  sulphuric  acid  and 
iodine.] 

For  the  detection  of  tannin,  a  very  dilute  solution 
of  chlor-iodide  of  zinc  is  employed,  the  contents 
of  cells  which  contain  tannin  becoming  reddish  or 
violet  under  this  treatment.2 

POTASSIC  HYDRATE  (Caustic  Potash),. 

Next  to  iodine,  caustic  potash  takes  the  most 
important  place  among  micro-chemical  reagents.3 

1  Schacht :  Die  Pflanzenzelle,  p.  143,  et  seq.     Hofmeister :  Handbuch, 
I.,   Die  Pflanzenzelle,  p.  258. 

2  Sanio:  Bot.  Zeit.,  1863.     Dippel.  Das  Mikroskop,  L,  p.  375. 

8  Nageli:  Das  Mikroskop,  1877,  pp.  472  and  525.  Dippel:  Das  Mik- 
roskop, I.,  p.  278.  Wiesner :  Technische  Mikroskopie,  1867.  Sachs: 
Ueber  die  Stoffe,  welche  das  Material  der  Zellhaute  liefern.  —  Jahrb.  fur. 
wiss.  Bot,  1863,  III.;  Keimung  von  Allium  Cepa.  —  Bot.  Zeitung,  1863, 
Nos.  8-9 ;  Zur  Keimungsgeschichte  der  Gra'ser,  Keimung  der  Dattel.  — 
Bot.  Zeitung,  1862. 


IO  MICRO-CHEMICAL  REAGENTS. 

The  commercial  potassic  hydrate  is  used  in  an 
aqueous  or  alcoholic  solution,  whose  concentra- 
tion depends  upon  the  particular  purpose  for  which 
it  is  to  be  employed.  In  general,  a  moderately 
concentrated  solution  is  preferable,  since  it  may 
be  diluted  when  this  is  necessary,  and  a  very 
strong  solution  is  seldom  to  be  recommended, 
since,  although  its  action  is  more  rapid,  it  destroys 
the  tissues  too  quickly.  In  making  an  aqueous 
solution,  care  should  be  taken  to  add  the  alkali  in 
small  quantities,  to  avoid  undue  heating.  The 
alcoholic  solution  is  made  as  follows : x  85-90  per 
cent,  alcohol  is  mixed  with  a  concentrated  aqueous 
solution  of  caustic  potash  until  a  precipitate  is 
formed.  After  being  repeatedly  shaken  it  is  set 
aside  for  twenty-four  hours,  at  the  end  of  which 
time  the  clear,  pale-yellow  fluid  is  decanted,  and, 
after  dilution  with  distilled  water,  is  ready  for  use. 
Russow  recommends  the  employment  of  solutions 
of  two  densities,  one  consisting  of  one  part  of  dis- 
tilled water  and  two  parts  of  the  saturated  solu- 
tion ;  the  other,  of  one  part  of  water  to  three  of 
the  original  solution. 

Since  potash  solutions  readily  take  up  carbonic 
acid  from  the  air,  and  further  tend  to  crystallize 
out  in  the  neck  of  the  bottle  in  which  they  are 
'kept,  it  is  necessary  to  keep  them  in  bottles  with 
glass  stoppers,  which  must  be  frequently  loosened. 

1  Russow:  Mem.  Acad.  St.  Petersbourg,  Ser.  7,  Tome  19,  No.  i,  p.  15, 
note. 


POTASSIC    HYDRATE.  II 

[A  slight  coating  of  paraffine  will  prevent  the 
stopper  from  sticking.] 

In  microscopic  manipulations  the  value  of  potash 
depends  upon  its  solvent  and  softening  actions, 
those  which  accompany  the  absorption  of  water. 
It  dissolves  many  of  the  fine  granules  in  proto- 
plasm, bleaches  various  coloring  matters,  forms 
soluble  soaps  with  fats,  and  effects  the  swelling  of 
the  starch  which  often  renders  tissues  opaque.  It 
thus  destroys  the  protoplasmic  structure  of  cells 
and  makes  these  clearer  and  more  transparent, 
thus,  when  dilute,  playing  a  very  important  part 
as  a  clearing  medium  in  the  study  of  otherwise 
opaque  sections  ;  while  it  permits  the  examination 
of  thick  masses  of  tissue,  or  even  of  entire  organs, 
as  embryos,  trichomes,  sections  of  the  punctum 
vegetationis,  or  whole  stems  and  leaves^  as  in  the 
study  of  the  course  of  fibre-vascular  bundles  [and 
laticiferous  tissue].1 

This  method  was  first  proposed  by  Hanstein. 
The  sections  are  treated  with  a  solution  of  potash, 
washed,  and  neutralized  with  hydrochloric  or  acetic 
acid.  If  this  renders  them  too  opaque  they  may  be 
cleared  by  washing  first  in  pure  water,  then  in 
ammonia  water.  If,  on  the  other  hand,  they  are 
too  transparent,  they  may  be  improved  by  washing 


1  Hanstein :  Die  Scheitelzellgruppe  im  Vegetationspunkt  der  Phanero- 
gamen,  1868;  Entwickelung  des  Keimes,  Botan.  Abhandl.,  L,  Heft  i,  p.  5. 
Koch :  Cuscuta,  in  Hanstein's  Bot.  Abhandl.,  II.,  Heft  3,  p.  25.  Dels : 
Anat.  d.  Droseraceen.,  Diss.,  Breslau,  1879,  P-  J3-' 


OF  THE 
MMIX/FRSITY 


12  MICRO-CHEMICAL  REAGENTS. 

in  alum  water.  Sometimes  the  process  has  to  be 
repeated  several  times  before  the  desired  effect  is 
obtained.  After  washing  in  distilled  water,  prep- 
arations made  in  this  way  can  be  kept  for  a  long 
time  in  glycerine,  which  clears  them  still  more. 

Russow's  alcoholic  potash  is  used  for  the  same 
purpose  as  the  aqueous  solution,  'and  in  most  cases 
is  preferable,  since  the  presence  of  alcohol  pre- 
vents the  excessive  swelling  of  the  cell-wall,  that/ 
often  occurs  when  the  Hanstein  process  is  em- 
ployed. With  this  reagent  acetic  acid  may  be 
used  for  neutralization,  and  the  preparations  keep 
well  in  glycerine.  Starch  swells  and  is  destroyed 
in  potash,  which  is  therefore  useful  for  clearing  up 
tissues,  like  the  albumen  of  seeds,  that  contain 
much  starch. 

The  aqueous  solution  of  potash,  causing  swelling 
of  the  cell-wall,  often  facilitates  the  study  of  its 
striation  and  stratification ;  this  is  especially  the 
case  in  collenchyma. 

A  warm  solution  of  potash,  being  a  solvent  for 
the  so-called  intercellular  substance,  is  sometimes 
employed  for  isolating  cells  by  maceration. 

Potash  is  useful  as  a  test  for  suberin.1  When 
thin  sections  of  corky  tissue  are  well  boiled  in  this 
reagent  the  suberin  is  extracted  from  the  cell- 
wall,  appearing  as  yellow  drops,  that  soon  run 
together. 

1  Hohnel:  Kork  u.  verkorkte  Gewebe.  —  Sitzber.  wien.  Akad.,  1877, 
Abth.  I.,  p.  1 6. 


POTASSIC   HYDRATE.  13 

Cell-walls  (wood-cells,  ducts,  etc.),  that  do  not 
immediately  give  the  cellulose  reaction  with  iodine 
and  sulphuric  acid,  because  of  the  presence  of 
so-called  incrustation  matters,  give  this  reaction 
promptly  after  treatment  with  boiling  potash1  — 
often  used  in  connection  with  nitric  or  other 
acids  —  since  this  removes  the  foreign  sub- 
stances. 

Sometimes  tannin  may  be  recognized  by  the 
use  of  potash,  as  cells  which  contain  it  —  and 
which  color  green  with  salts  of  iron  —  assume  a 
yellow  color  with  potash. 

Cells  containing  chrysophanic  acid2  become  pur- 
ple-red with  the  same  fluid. 

Sachs  has  employed  potash  as  an  analytic  re- 
agent in  various  histologo-physiological  studies, 
obtaining  good  reactions  when  using  it  with  cupric 
sulphate  in  testing  for  different  sugars,  protein 
matters,  and  carbo-hydrates.3 

When  protoplasm  is  first  treated  with  nitric  acid, 
and  afterwards  with  dilute  potash  or  ammonia,  it 
assumes  a  beautiful  yellow  color  from  the  forma- 
tion of  potassic  or  ammonic  xanthoproteate.4 

The  so-called  crystalloids  swell  and  change  their 
angles  in  potash,  thus  showing  their  organic 
nature. 

1  Hofmeister :  Handbuch,  I.,  several  places. 

2  Borscow :  Bot.  Zeit,  1874,  p.  20.     Weiss  :  Allg.  Bot.,  1878,  I.,  p.  288. 
*  See  cupric  sulphate,  below.     The  reader  is  referred  to  the  preceding 

papers,  and  to  H.  de  Vries'  Keimung  des  rothen  Klees.  —  Landwirthsch. 
Jahrb.,  1877,  VI.,  p.  468. 

4  Dippel :  Das  Mikroskop,  II.,  pp.  10, 18. 


14  MICRO-CHEMICAL  REAGENTS. 

AMMONIA. 

A  concentrated  solution  of  ammonia  in  water  is 
often  used  instead  of  potash,  where  the  latter 
would  act  too  powerfully.1  We  have  also  men- 
tioned its  use  in  clearing  tissues  by  Hanstein's 
method. 

As  a  test  for  protein  combinations,  including 
crystalloids,  it  is  employed  with  nitric  acid  to  in- 
tensify the  color  of  the  xantho-protein  reaction. 
When  a  thin  section  of  a  tissue  composed  of  cells 
with  thickened  walls  is  successively  treated  with 
nitric  acid  and  ammonia,  the  middle  lamella  (inter- 
cellular substance)  is  colored  yellow.2 

Finally,  it  is  employed  in  the  preparation  of 
cupro-sulphate  and  carminate  of  ammonia.3 

Ammonia  is  also  valuable  for  restoring  the  form 
of  herbarium  specimens  of  phaenogams,  algae,  and 
mosses,  as  well  as  spores,  pollen  grains,  etc.,  that 
are  to  be  examined  microscopically. 

CUPRAMMONIA  (Cuoxam,  Cramer;  Cupridiamin). 

This  important  reagent  must  be  freshly  made 
when  needed  for  use,  as  it  deteriorates  with  age. 
When  it  is  desirable  to  keep  it  for  a  time  it  should 
be  set  in  the  dark. 

1  Dippel:    Das  Mikroskop,  L,  p.  279.     Weiss:   Allgem.  Bot.,  1878, 
pp.  77,  144. 

2  Dippel:  Das  Mikroskop,  II.,  p.  100. 
8  See  the  section  on  staining  agents. 


CUPRAMMONIA.  15 

To  prepare  it  an  aqueous  solution  of  sodic 
hydrate  is  slowly  added  to  a  solution  of  cupric  sul- 
phate, until  a  precipitate  of  cupric  hydrate  forms. 
The  precipitate  is  collected  on  a  filter,  transferred 
to  a  test  tube,  washed,  and  dissolved  in  strong 
ammonia.  The  solution,  which  is  of  a  beautiful 
dark-blue  color,  is  at  once  ready  for  use. 

It  may  also  be  prepared  by  allowing  16  per  cent, 
ammonia  to  stand  upon  copper  turnings  in  an  open 
flask.  However  prepared,  it  should  be  used  only 
so  long  as  it  has  the  power  of  quickly  dissolving 
cotton  fibers. 

Pure  cellulose  swells  much  and  is  dissolved  with- 
out conversion  into  amyloid.  Cell-walls  incrusted 
with  lignin,  subenn,  etc.,  are  only  dissolved  after 
these  substances  have  been  removed  by  Schultze's 
maceration. 

In  general,  neither  the  cuticle  nor  the  so-called 
middle  lamella  or  intercellular  substance  dis- 
solves. 

According  to  Kabsch,  cuprammonia  is  a  test  for 
pectose.  When  a  tissue  containing  this  substance 
is  treated  with  the  reagent  a  fine  skeleton  of  cupric 
pectate  is  left  behind.1 

1  Schweitzer:  Vierteljahrschrift,  nat.  Ges.,  Zurich,  1857,  II.  Kabsch: 
Jahrb.  wiss.  Bot.,  1863,  p.  357.  Hofmeister :  Handbuch,  I.,  p.  240. 
Dippel :  Das  Mikroskop,  I.,  p.  280.  Wiesner :  Technische  Mikroskopie, 
1867,  p.  38.  note  2.  Nageli:  Das  Mikroskop,  1877,  p.  474.  Fremy: 
Mem.  de  1'  Acad.,  Paris,  1859;  Journ.  de  Pharm.  et  Chim.,  XXXVI.  Ep- 
stein :  De  Conjunctione  cellulosse  cum  cupro  oxydato.  Dissertatio,  Bres- 
lau,  1860  (Cf,  Bot.  Zeit,  1860,  p.  234). 


1 6  MICRO-CHEMICAL  REAGENTS. 


MINERAL    ACIDS.1 


SULPHURIC    ACID  (Oil  of  Vitriol). 

Sulphuric  acid  is  used  in  a  concentrated  or  dilute 
form  according  to  circumstances.  The  most  use- 
ful proportion  is  obtained  by  diluting  one  volume 
of  strong  acid  with  three  of  water.2 

Dilute  sulphuric  acid  causes  starch  grains  to 
swell,  and  similarly  affects  cellulose,  especially  in 
collenchyma,  at  the  same  time  transforming  it 
into  one  of  its  isomers,  amyloid,  which  differs  from 
cellulose  in  assuming  a  blue  color  when  treated 
with  iodine.  Hence,  to  determine  whether  a  cell- 
wall  consists  of  pure  cellulose,  it  is  only  necessary 
to  treat  it  first  with  a  tincture  of  iodine  and  then 
with  sulphuric  acid,3  when  it  turns  blue,  if  unin- 
crusted. 

Concentrated  sulphuric  acid  dissolves  both  cell- 
wall  and  starch  grains,  greatly  swelling  all  parts  it 


1  In  using  any  of  these  acids  a  large  cover-glass  should  be  employed  to 
prevent  injury  to  the  objective. 

2  Dippel:  Das  Mikroskop,  L,  p.  276.     Na'geli:   Das  Mikroskop,  1877, 
p.  474.     Weiss:   Allgem.  Bot,  1878,  L,  p.  62,  etc.      Hofmeister:    Hand- 
buch,  L,  p.  252,  etc. 

8  [M.  Vetillart  advises  the  use  of  the  following  mixture,  in  place  of  pure 
acid,  in  the  cellulose  test.  Three  volumes  of  sulphuric  acid  (spec.  grav. 
1.84),  one  of  water,  and  two  of  glycerine,  are  slowly  mixed  to  avoid  heat- 
ing. This  does  not  destroy  the  tissue.  —  Christy's  Fibres,  p.  17.  w.  T.] 


MINERAL   ACIDS.  I/ 

comes  in  contact  with.     Starch  is  thus  converted 
into  dextrin. 

The  cuticularized  parts  of  the  cell-wall  (cork, 
cuticle,1  extine  of  pollen,  exospore,  root-cap,  etc.) 
resist  the  action  of  this  reagent,  as  does  the  pre- 
viously mentioned  middle  lamella.  Protoplasm  is 
destroyed  after  a  time,  while  young  protoplasmic 
bodies  are  often  colored  rose-red  under  its  action,2 
the  reaction  being  rendered  more  certain  by  the 
addition  of  a  solution  of  cane-sugar.  Fat-bodies 
occurring  in  the  protoplasm  are  not  dissolved,  but 
run  together,  forming  small  refractive  drops.  Oil 
which  previously  existed,  diffused  through  the  pro- 
toplasmic mass,  manifests  itself  similarly.3 


NITRIC  ACID   (Aqua  fortis). 

This  is  employed  as  a  macerating  reagent  in 
combination  with  potassium  chlorate,4  q.  v. 

When  the  contents  of  a  cell  are  treated  with 
nitric  acid  alone,  or  with  this  reagent  followed  by 
ammonia,  they  assume  a  bright  yellow  color  when 
protein  matters  are  present,  through  the  formation 
of  xantho-protein  acid. 

According  to  Hb'hnel,  nitric  acid,  either  alone  or 

1  DeBary:   Hofmeister's   Handbuch,  III., -1878,  pp.  84,  131.      Bron- 
gniart :  Ann.  Sci.  Nat.,  1830,  i  Ser.,  T.  XXL,  p.  427. 

2  Sachs:  Bot.  Zeitung,  1862,  p.  242. 

3  Sachs:  Bot.  Zeitung,  1862,  p.  146. 

4  Dippel:  Das  Mikroskop,  I.,  p.  275.     Nageli:  Das  Mikroskop,  1877, 
p.  474.    Sanio :  Bot.  Zeit.,  1863,  p.  362,  note. 


1 8  MICRO-CHEMICAL  REAGENTS. 

with  potassic  chlorate,  is  a  good  test  for  suberin.1 
To  obtain  this  so-called  eerie  reaction,  thin  sec- 
tions of  the  tissue  are  treated  with  the  reagent. 
If  suberin  is  present,  after  the  solution  of  the 
other  parts  of  the  cell-wall,  yellow  or  spherical 
masses  remain,  which  are  at  first  granular  but 
later  become  homogeneous.  They  consist  of  eerie 
acid,  and  are  soluble  in  alcohol,  ether,  benzol,  and 
chloroform. 

Warm  nitric  acid,  followed  by  ammonia,  colors 
the  middle  lamella  (or  intercellular  substance) 
yellow.2 

Nitric  acid  dissolves  starch  grains  after  causing 
them  to  swell  greatly ;  hence  it  may  often  be  used 
in  a  dilute  condition  for  clearing  tissues  which 
contain  much  starch. 


CHROMIC  ACID. 

This  must  be  free  from  sulphuric  acid.  It  is 
employed  concentrated  or  dilute,  according  to  cir- 
cumstances.3 Since  it  tends  to  form  crystals  in 
the  neck  of  the  bottle  in  which  it  is  kept,  the 
stopper  should  be  coated  with  vaseline,  or  fre- 
quently loosened  by  turning. 

The  cell-wall  swells  and  finally  dissolves  in  chro- 

1  Hohnel :  Sitzber.   wien.  Akad.,  1877,  Abth.  I. 

2  Dippel :  Das  Mikroskop,  II.,  p.  100. 

3  Dippel:  Struktur  der  Zellhiille,  1878.     Sanio :  Bot.  Zeitung,  1860- 
1863.    Kabsch:  Jahrb.  wiss.  Bot.,  III.,  p.  387.     Nageli :   Das  Mikroskop, 
1877,  P-  475- 


MINERAL  ACIDS.  19 

mic  acid,  and,  as  the  swelling  proceeds  slowly,  the 
dilute  acid  is  useful  for  showing  the  stratification  of 
the  wall.  Only  silicified  and  corky1  layers  resist 
its  action.  Lignified  cells  are  entirely  dissolved. 
Those  containing  suberin  become  very  transparent 
and  nearly  invisible ;  but  after  the  acid  has  been 
removed  by  washing  they  usually  reappear,  though 
the  prolonged  action  of  the  reagent  dissolves 
them. 

In  general,  chromic  acid  is  useful  in  the  study 
of  the  stratification  of  the  cell-wall,  starch-grains, 
etc.  It  is  also  sometimes  applicable  to  the  fixation 
of  protoplasm  \e.  g.  the  plasmodia  of  Myxomy- 
cetes],  but  must  be  used  in  a  very  dilute  form  for 
this  purpose. 


'  PEROSMIC  ACID. 

This  poisonous  and  ill-smelling  reagent,2  which 
is  usually  kept  in  the  crystalline  form  in  hermet- 
ically-sealed tubes,  and  dissolved  in  water  when 
needed  for  use,  has  of  late  years  been  much  em- 
ployed in  the  investigation  of  the  minute  structure 


1  Hohnel:   Ueber  Kork.  —  Sitzungsber.  wiener  Akad.,  1871,  Abth.,  I. 
Hohnel  differs  here  from  Pollender :    Chromsaure  als  Losungsmittel  fur 
Pollenin  und  Cutin.  —  Bot.  Zeitung,  1862,  p.  385. 

2  Dippel:   Das  Mikroskop,  L,  p.  375.     Strasburger :  Befruchtung  und 
Zelltheilung,  1878;  Studien  iiber  Protoplasma,  1876.     Pfitzer:  Die  Bacil- 
lariaceen.  —  Hanstein's  Bot.  Abhandl.,  I.,  Heft  2,  1871,  p.  33.    Nageli :  Das 
Mikroskop,  1877,  P-  476.     Ranvier :    Histologie,  1875,  p.   55.      Robin: 
Microscope,  1877,  P«  22°- 


2O  MICRO-CHEMICAL  REAGENTS. 

of  protoplasm.  As  its  vapor  attacks  the  mucous 
membrane  of  the  eyes  and  air  passages,  the  use  of 
the  osmiamid  has  been  recommended,  instead  of 
the  acid,  which  it  replaces  well  in  all  reactions. 
Oil  and  fats  reduce  osmic  acid,  precipitating  me- 
tallic osmium,  which  colors  the  oil  drops  brown  or 
even  black.  Tannin  is  recognizable  by  the  same 
reaction. 

A  one  per  cent,  solution  is  especially  valuable  for 
the  instantaneous  hardening  of  living  protoplasm. 
The  stages  in  the  division  of  the  nucleus  of  cells, 
and  other  structural  peculiarities  of  protoplasmic 
bodies  are  thus  fixed  in  a  few  minutes,  and,  after 
washing,  may  be  preserved  for  future  study  in  a 
dilute  solution  of  glycerine,  which,  however,  has 
the  disadvantage  of  rendering  the  preparations 
very  transparent. 

Recently  perosmic  acid  has  been  used  in  the 
study  of  young  meristem  tissues.1  The  organs  to 
be  studied  were  laid  in  a  very  dilute  aqueous  solu- 
tion (.1-1  per  cent.)  of  the  acid  until  they  black- 
ened, after  which  they  were  treated  with  alcohol, 
cleared  with  clove  oil,  and  imbedded  for  sectioning 
in  cocoa-butter  (butyrum  cacao  of  pharmacists). 
The  sections  were  mounted  in  Canada  balsam. 

A  mixture  of  nine  parts  of  .25  per  cent,  chro- 
mic acid  and  one  part  i  per  cent,  perosmic  acid 


1  Parker:  Journ.  Roy.  Microsc.  Soc.,  1879,  ^v  P-  3%2'     I  have  not 
tested  the  method. 


MINERAL   ACIDS.  21 

also  gave  good  results.  Staining  and  hardening 
were  thus  effected  simultaneously,  the  terminal 
buds  of  Chara  serving  as  material  for  the  study. 


PHOSPHORIC   ACID. 

This  has  a  limited  usefulness  through  inducing 
the  imbibition  of  water.  It  causes  crystalloids  to 
swell.1  [Its  occasional  use  in  the  cellulose  test 
has  already  been  mentioned  cf.  p.  5.] 


HYDROCHLORIC  ACID  (Muriatic  Acid). 

Like  other  powerful  acids  this2  induces  the 
swelling  of  starch,  and  young  cell-walls,  especially 
when  it  is  concentrated.  Its  employment  in  Han- 
stein's  method  of  clearing  tissues  has  been  already 
mentioned  (cf.  p.  n).  Kabsch3  has  used  it  with 
concentrated  sulphuric  acid  and  potash  to  isolate 
the  tertiary  lamella  of  wood-cells,  the  sections  be- 
ing successively  treated  with  the  separate  reagents, 
and  washed  with  water  after  each  has  acted  a  suf- 
ficient length  of  time. 

After  lying  in  the  acid  for  a  long  time  nitrogen- 
ous substances  (protein  matters)  assume  a  violet 


1  Hofmeister:   Handbuch,  I.,  several  places.      Kraus:    Jahrb.    wiss. 
Bot.,  VIII.,  Crystalloids  in  the  Epidermis. 

2  Dippel:   Das  Mikroskop,  I.rp.  276;    II.,  p.  41.     Hdhnel:  Sitzber. 
wien.  Akad.,  1877,  Abth.,  I.,  p.  21.  , 

3  Jahrb.   wiss.  Bot.,  1863,  III. 


OF  THE 

UNIVERSITY 

OF 


22  MICRO-CHEMICAL  REAGENTS. 

color.  Hydrochloric  acid  is  further  valuable  for 
showing  the  nucleus  of  diatoms,  etc.1 

Crystals  based  on  carbonic  acid  emit  bubbles  of 
this  gas,  when  treated  with  hydrochloric  acid,  the 
carbonates  being  converted  into  chlorides.  On 
the  other  hand,  crystals  based  on  oxalic  acid  dis- 
solve without  effervescence. 

Recently,  Pringsheim2  has  employed  hydro- 
chloric acid  as  a  reagent  for  hypochlorin,  one  of 
the  components  of  chlorophyll  bodies,  q.  v.  Newly- 
cut  sections  are  allowed  to  lie  in  the  acid  for  sev- 
eral hours,  when  the  hypochlorin  separates  as 
small  semi-fluid  exudation-masses,  brownish  or  red 
in  color,  at  first  nearly  spherical,  but  afterward 
forming  needle-shaped  crystals. 


ORGANIC   ACIDS. 


ACETIC   ACID. 

This  acid 3  is  used,  in  the  form  in  which  it  is  kept 
by  pharmacists,  in  various  micro-chemical  investi- 
gations; e.g.  it  may  replace  hydrochloric  acid  in 
Hanstein's  method  for  clearing  opaque  meristem 
(see  the  section  on  potassic  hydrate,  Cf.  p.  1 1).  The 
sections,  first  rinsed  in  water,  are  placed  in  a  drop 

1  Pfitzer :  Bacillariaceen.  —  Hanstein's  Bot.  Abhandl.,  I.,  Heft  2,  p.  31. 

2  Monatsber.  berlin.  Akad.,  Nov.  1879. 

3  Dippel:  Das  Mikroskop,  L,  p.  277;  II.,  p.  IQ.     Nageli:  Das  Mikro- 
skop,  1877,  p.  476.    Hanstein:  Scheitelzellgruppe,  1868;  Entwickelung 
des  Keimes.  — Bot.  Abh.,  Bd.  L,  Heft  i,  p.  5.    Strasburger:  Studien 
ttber  Protoplasma,  1876,  p.  5. 


ORGANIC   ACIDS.  23 

of  acetic  acid  on  the  slide.  With  the  neutraliza- 
tion of  the  alkali  they  often  become  somewhat 
opaque,  but  may  be  re-cleared,  often  to  great  trans- 
parency, by  laying  them  in  glycerine. 

In  the  study  of  crystals,  those  composed  of  an 
oxalate  may  be  distinguished  by  being  insoluble  in 
acetic  acid,  but  soluble  in  hydrochloric  acid, 
(p.  22)  ;  while  salts  of  carbonic  acid,  occuring  as 
crystals  or  as  incrusting  components  of  the  cell- 
wall,  are  soluble  with  effervescence  in  either. 

Acetic  acid  sharply  differentiates  the  nucleus, 
and  is  often  a  valuable  medium  in  the  study  of 
the  intimate  structure  of  protoplasm.  Stras- 
burger1  employs  a  one  per  cent,  aqueous  solution 
for  fixing  the  nucleus  when  staining  the  latter 
with  methyl-green ;  at  the  same  time  it  often 
clears  up  protoplasmic  structures,  and  swells  the 
condensed  ectoplasm. 

Acetic  acid  has  also  found  application  in  coch- 
ineal solution,  and  in  glycerine  in  which  prep- 
arations stained  with  carmine  are  to  be  preserved. 


OXALIC  ACID. 

The  aqueous  solution2  is  employed  with  certain 
coloring  matters  in  staining  tissues.    The  alcoholic 


1  Zellbiid.u.  Zelltheil.,  1880,  3  ed. 

2  Dippel:  Das  Mikroskop,  I.,  p.  285.     Frey:  Das  Mikroskop,  p.  77. 
Bachmann:  Dauerpraparate,  p.  27.     Wiesner:  Technische  Mikroskopie, 
pp.  247,  258. 


24  MICRO-CHEMICAL  REAGENTS. 

solution  is  useful  for  removing  some  of  the  color 
from  too  deeply  stained  sections.  A  concentrated 
aqueous  solution  is  used  as  a  test  for  pectose, 
which  it  dissolves  after  previous  treatment  of  the 
section  with  potash. 


CARBOLIC  ACID  (Phenol). 

This  has  a  very  limited  application  in,  micro- 
chemistry.1  Cell-walls  that,  after  treatment  with 
carbolic  acid,  assume  a  greenish-yellow  or  bluish- 
green  color,  when  moistened  with  hydrochloric 
acid,  are  considered  to  be  lignified,  and  phenol 
thus  becomes  a  reagent  for  lignin  (?). 

Leitgeb2  has  employed  a  solution  of  carbolic 
acid  in  alcohol  as  a  clearing  medium  in  studying 
the  histogeny  of  mosses. 

Phenol  should  be  added  in  small  quantity  to 
glycerine-jelly  to  prevent  molding,  and  when  dilute 
it  may  be  used  as  a  preservative  for  bacteria 
(Warming),  which  remain  sharply  denned,  but  be- 
come clear  and  homogeneous  internally. 

1  Bot.  Zeitung,  1877,  P-  7§6. 

2  Nageli:  Das  Mikroskop,  1877,  p.  476. 


ALCOHOLS.  25 

ALCOHOLS. 


ALCOHOL  (Ethyl  Alcohol). 

The  known  disinfecting  value  of  spirits  of  wine, 
or  alcohol,1  depends  upon  its  fatal  action  on  all 
protoplasm ;  hence  its  application  as  a  preservative 
for  animal  and  vegetable  preparations.  Absolute 
alcohol  has  the  property,  in  common  with  peros- 
mic  acid,  of  rendering  protoplasm  rigid,  and  it  is 
thus  applicable  in  studying  the  more  intimate 
structure  .of  protoplasmic  bodies,  the  division  of 
the  nucleus,  etc.  Its  avidity  for  water  causes  the 
protoplasm  to  contract  from  the  cell-wall,  so  that 
the  ectoplasm  becomes  visible ;  and  the  same 
peculiarity  is  taken  advantage  of  when  we  employ 
alcohol  for  hardening  tissues  that  are  too  soft  for 
section-cutting  when  fresh.  It  may  also  be  used 
advantageously  for  removing  the  air  from  intercel- 
lular spaces,  etc.,  in  preparations,  since  it  pene- 
trates into  capillary  cavities  much  more  readily 
than  water  does.  In  difficult  cases  warming  the 
sections  often  helps  this  action,  alcohol  being 
added  from  time  to  time  to  replace  that  lost  by 

1  Dippel:  Das  Mikroskop,  I.,  p.  282.  DeBary:  Vergl.  Anat.,  p.  86. 
Nageli:  Das  Mikroskop,  1877,  P-  476-  Sachs:  Bot.  Zeit,  1864,  Nos.  12- 
13.  Weiss:  Allg.  Botanik,  1878,  pp.  182,  185.  Tangl:  Protoplasma  der 
Erbse.- Sitzber.  wien.  Akad.,  Abth.  i,  1877-8.  Strasburger  :  Zellbildung 
und  Zelltheilung,  1875,  P-  2>  Befruchtung  und  Zelltheilung,  1878,  p.  38.^ 


.26  MICRO-CHEMICAL  REAGENTS. 

evaporation.  When  this  expedient  does  not  pro- 
duce the  desired  effect,  recourse  should  be  had  to 
the  air-pump. 

Alcohol  is  a  solvent  for  volatile  oils  and  resins, 
while  fatty  oils  and  vegetable  wax  are  insoluble  in 
cold  alcohol,  though  it  causes  the  oil  globules  to 
become  confluent. 

In  tissues,  e.  g.  nectaries,  which  contain  much 
cane  sugar,  this  may  be  forced  by  the  use  of  abso- 
lute alcohol  to  separate  in  small  stellate  crystals 
soluble  in  water.1 

In  all  tissues  containing  inulin  the  prolonged 
action  of  alcohol  effects  its  precipitation  within 
the  cell  in  the  form  of  sphaero-crystals ;  e.  g.  in 
Inula,  Helianthus,  Dahlia,  etc.  Other  sphaero- 
crystal-forming  substances,  such  as  hesperidin,2 
crystallize  under  the  same  treatment.  (See  Inulin 
and  Hesperidin.) 

Asparagin  may  also  be  detected  by  the  use  of 
absolute  alcohol,  the  sections  that  are  tested  being 
alternately  moistened  with  the  reagent  and  allowed 
to  dry,  when  the  asparagin 3  crystallizes  out  —  often 
with  other  substances.  It  is  recognized  by  its  in- 

1  Bonnier :  Les  Nectaires.  —  Ann.  Sc.  Nat.,  1879,  T.  8,  pi.  8,  figs.  124, 126. 

2  Prantl:  Das  Inulin,  1870.     Rosenvinge:  Sfserokrystaller  hos  Mesem- 
brianthemum.  —  Nat.   Foren.   vidsk.  Meddelelser,    1877-78,   p.   305,   with 
table  (with  literature  of  the  subject).      Pfeffer :    Sachs'  Lehrbuch,  1874,  P- 
65.     Russow:  Leitbiindelkryptogamen,  1872,  p.  no. 

3  Hartig:  Entwickelung  des  Pflanzenkeimes,  1858.     Pfeffer:  Ann.  Sci. 
Nat.,  Bot,  5  Sen,  T.  XIX.,  p.  391.     Sachs:   Lehrbuch  der  Botanik,  1874, 
p.  689.     Borodin:  Bot.  Zeitung,  1878,  p.  803.     Detmer:  Vergl.  Physiol.  d. 
Keimungsprocesses,  1880,  p.  171. 


ALCOHOLS.  27 

solubility  in  a  warm  solution  of  asparagin.  The 
same  treatment  may,  perhaps,  be  used  with  effect 
ia  testing  for  other  substances ;  it  has  already 
been  found  satisfactory  for  tyrosin. 

Alcohol  is  also  employed  as  a  solvent  for  a 
part  of  the  reagents  used  in  micro-chemical  tests ; 
e.g.  anilin  dyes,  corrosive  sublimate,  phloroglucin, 
iodine,  etc.,  as  well  as  in  the  capacity  of  an  anhy- 
drating  medium  for  preparations  that  are  to  be 
mounted  in  volatile  oils  or  Canada  balsam. 


GLYCERINE. 

This  fluid,1  especially  useful  as  a  preservative 
for  permanent  preparations,  for  which  use  I  pre- 
fer the  nearly  anhydrous  form  known  as  glyce- 
rinum  Wilsoni,  is  also  employed  for  many  other 
purposes.  According  to  circumstances  it  is  diluted 
with  alcohol  or  water,  or  with  both.  Preparatory 
to  final  mounting  it  is  often  well  to  place  prepara- 
tions temporarily  in  a  mixture  of  equal  volumes  of 
glycerine,  distilled  water,  and  absolute  alcohol. 

It  is  used  like  alcohol  as  an  anhydrating  medium 
in  the  study  of  protoplasm.  It  can  be  employed 
very  successfully  as  a  clearing  medium  in  many 
cases  ;  e.  g.  in  studying  the  histology  of  the  fibro- 
vascular  bundles,  and  as  a  preservative  or  final 

1  Kraus:  Bot.  Zeitung,  1877,  p.  329.  Sachs:  Bot.  Zeitung,  1864,  Nos. 
12,13.  Nageli:  Das  Mikroskop,  1877,  p.  475.  Hegelmaier:  Entwick- 
elung  dicotyledoner  Keime,  p.  n. 


28  MICRO-CHEMICAL  REAGENTS. 

clearing  fluid  in  the  Hanstein  and  Russow  meth- 
ods of  clearing  tissues.  A  mixture  of  dilute  potash 
and  glycerine  has  been  employed  by  Hegel maier  in 
the  study  of  the  embryo.1 

Kraus  has  employed  glycerine  as  a  reagent  for 
sugar  and  inulin.  When  sections  that  contain 
these  substances  in  solution  are  placed  in  glyce- 
rine, strongly  refractive  rounded  drops  appear  in 
the  cells.  If  .inulin  is  present  these  drops  change 
to  the  characteristic  spaero-crystals,  and  remain ; 
but  if  only  sugar  is  present  in  the  tissue  they 
rapidly  dissolve  again.  The  sections  should  not 
be  laid  in  water,  but  must  be  placed  directly  in  the 
glycerine.  The  reliability  of  this  reaction  is  cer- 
tainly worthy  of  further  tests.  With  larger  masses 
of  tissue,  glycerine  can  also  be  used  for  the  sepa- 
ration of  inulin,  and  it  is  a  good  preservative 
medium  for  inulin  preparations. 

lodin-glycerine  is  useful  in  the  study  of  protein 
grains.  It  is  prepared  by  dissolving  a  little  iodine 
in  glycerine,  to  which  a  small  quantity  of  iodide  of 
potassium  has  previously  been  added.  More  spe- 
cific directions  as  to  the  relative  quantities  are  un- 
necessary. 

Warm  glycerine  is  also  used  for  the  same  pur- 
pose. In  it  the  protein  grains,  which  under  natural 
conditions  are  uniformly  refractive,  become  differ- 

1  [For  the  use  of  glycerine  in  the  cellulose  test,  see  sulphuric  acid,  p.  16. 
For  its  employment  instead  of  potassium  tartrate  in  Fehling's  sugar  test, 
see  cupric  sulphate,  p.  36.  w.  T.] 


ETHERS,   ETC.  2Q 

entiated  so  that  globoids  and  crystalloids  are  dis- 
tinguishable. 

Since  the  evaporation  of  glycerine  is  almost 
imperceptible,  it  is  one  of  the  best  preservatives 
for  permanent  preparations ;  but  as  it  absorbs  the 
moisture  of  the  air  when  concentrated,  the  cover- 
glass  should  be  sealed  with  some  air-tight  cement. 


ETHERS,  ETC. 


ETHER,  BENZOL,  CHLOROFORM,  BISULPHIDE   OF 
CARBON,  ETHEREAL-OILS.1 

As  reagents  for  determining  to  what  extent  a 
substance  is  a  fat,  a  volatile  oil,  a  resin,  etc.,  sub- 
stances have  been  employed  which,  from  a  chemi- 
cal standpoint,  are  often  quite  different.  They 
allow  us  to  recognize  these  substances  because  of 
their  solvent  powers.  For  this  purpose  ether, 
chloroform,  alcohol,  benzol,  oil  of  turpentine,  and 
carbon  bisulphide  are  used. 


l  Weiss:  Allgemeine  Bot,  1878,  p.  178.  DeBary :  Vergl.  Anatomic, 
p.  86.  H.  de  Vries :  Keimung  des  rothen  Klees.  —  Landwirthschaftliche 
Jahrb.,  1877,  Bd.  VI.,  p.  468.  Nageli :  Das  Mikroskop,  1877,  p.  476. 
Dippel :  Das  Mikroskop,  I.,  p.  374.  Wiesner :  Technische  Mikroskopie, 
p.  8 1.  For  the  use  of  carbon  bisulphide  as  a  reagent  for  sulphur,  see  this 
substance  in  Part  II. 


3O  MICRO-CHEMICAL  REAGENTS. 

Resins  are  soluble  in  ether,  cold  absolute  alco- 
hol, carbon  bisulphide,  and  oil  of  turpentine. 

Fatty  oils  are  soluble  in  carbon  bisulphide,  ethe- 
real oils,  hot  alcohol,  and  ether.  When  treated 
with  concentrated  potassic  hydrate  they  form 
soaps,  which  are  soluble  in  water. 

Ethereal  oils  are  easily  soluble  in  oil  of  turpen- 
tine and  cold  absolute  alcohol.  Most  of  them  are 
also  dissolved  by  ether  and  carbon  bisulphide.1 

It  will  be  seen  that  their  behavior,  when  treated 
with  alcohol,  is  distinctive  for  fatty  and  volatile 
oils ;  but  for  resins  other  reactions  must  be  relied 
upon,  which  will  be  spoken  of  later. 

The  substances  which  dissolve  fatty  oils  may  also 
be  used  as  reagents  for  wax. 

With  respect  to  the  application  of  these  reagents, 
we  can  only  say  that  since  carbon  bisulphide,  ether, 
oil  of  turpentine,  and  benzol  are  insoluble  in  water, 
the  sections  should  be  placed  immediately  in  them 
to  secure  the  best  results.  They  cannot  be  pre- 
pared in  water  which  is  replaced  by  allowing  the 
reagent  to  penetrate  under  the  cover-glass,  as  with 
so  many  other  reagents.  The  most  convenient 
plan  is  to  treat  the  sections  in  a  watch-glass,  with 
a  considerable  quantity  of  the  reagent. 

1  Dippel  (Mikr.,  I.,  p.  374)  states  that  they  are  insoluble  in  ether, 
which  I  do  not  understand. 


INORGANIC    SALTS.  31 


ETHEREAL  OILS. 

Several  volatile  oils  beside  turpentine,  which  has 
already  been  mentioned,  find  use  in  microscopy. 
Oil  of  cloves  and  lemon  oil  are  especially  useful  as 
clearing  fluids  in  the  study  of  pollen.  They  are 
also  good  preservative  media  for  objects  which  can- 
not be  studied  in  water,  but  require  a  fluid  of  some 
other  refractive  index.  Since  they  decrease  the  re- 
fraction they  are  very  useful  in  the  study  of  many 
strongly  refractive  substances,  by  the  aid  of  polar- 
ized light.  Preparations  which  have  been  in  oil 
must  be  washed  with  ether  or  chloroform  and 
afterward  with  alcohol  before  they  can  be  placed 
in  water  or  glycerine. 


INORGANIC   SALTS. 


CHLORIDE  OF  SODIUM  (Table  Salt). 
A  dilute  aqueous  solution1  is  used  as  a  morpho- 
logical reagent  for  the  contraction  of  protoplasmic 
bodies,  a  phenomenon  which  is  to  be  attributed  to 
its  avidity  for  water.  Many  other  salt-solutions 
have  this  property.  A  dilute  solution  of  table  salt 

1  Dippel :  Das  Mikroskop,  L,  p.  279. 


32  MICRO-CHEMICAL    REAGENTS. 

has  the  power  of  dissolving  crystalloids,  at  least  in 
the  embryo  of  Bertholletia.1 


CHLORIDE   OF  CALCIUM. 

In  an  aqueous  solution  (two  to  three  parts  of 
water  to  one  of  the  salt)  this  substance  is  some- 
times used  as  a  mounting  medium  for  permanent 
preparations  excepting  those  which  contain  amy- 
lum,  although  for  this  purpose  glycerine  has  largely 
replaced  it.  Recently  it  has  found  application  for 
clearing  tissues.  The  section  which  is  to  be  treated 
is  placed  in  a  few  drops  of  water  and  sprinkled  with 
the  dry  pulverized  salt.  It  is  then  warmed  over  a 
gentle  flame  until  nearly  dry,  and  again  moistened 
with  a  few  drops  of  water,  after  which  it  is  laid  in 
glycerine,  where,  in  the  course  of  a  few  hours,  it 
acquires  a  very  satisfactory  degree  of  transparency. 
(Treub's  method.2) 


CHLORIDE   OF  MERCURY  (Corrosive  Sublimate). 

A  very  dilute  aqueous  solution  (1:100)  is  used  to 
make  the  finest  protoplasmic  currents  evident.3 


1  Weyl :  Zeitschr.  fur  phys.  Chemie,  Bd.  I.,  p.  90. 

2  Treub:   Meristeme  primitif  de  la  racine.  —  Muse"e  de  Leyde,  1876, 
Tome  II.,  p.  9.      Eriksson :   Meristemet  i  dicotyla  vaxters  rotter,  p.  10. 
Flahault :  Accroissement  terminale  de  la  racine.  —  Ann.  des  Sc.  Nat.,  Bot., 
1878,  p.  24. 

3  Dippel :  Das  Mikroskop  I.,  p.  281. 


INORGANIC  SALTS.  33 

Pf eff er r  has  used  it  in  a  two  per  cent,  alcoholic 
tincture  in  the  study  of  protein  grains.  It  unites 
with  these  albuminoids,  forming  a  compound  insol- 
uble in  water ;  but  to  secure  satisfactory  results 
the  preparation  must  lie  at  least  twelve  hours  in 
the  fluid. 

CHLORIDE  OF  IRON. 

An  aqueous  solution  may  be  employed  as  reagent 
for  tannin2  when  this  is  not  present  in  too  small 
quantity.  The  cells  to  be  examined,  when  placed 
immediately  in  the  reagent  without  the  previous 
contact  of  water,  which  easily  removes  the  tannin, 
assume  a  dark  green  or  bluish-black  color,  accord- 
ing to  the  nature  of  the  tannin  compound.  The 
green  cells  color  yellow  if  potash  is  added. 

The  solution  should  not  be  too  concentrated,  as 
the  tannate  of  iron  which  is  formed  is  soluble  in 
an  excess  of  this  compound,  and  its  prompt  solu- 
tion renders  the  test  less  evident.  For  this  reason 
acetate  and  sulphate  of  iron3  have  largely  replaced 
the  chloride.  Being  more  certain  in  their  action, 
they  are  to  be  preferred. 

1  Pfeffer :  Jahrb.  fur  wiss.  Bot.,  1872,  VIIL,  p.  491.     Weiss :  Allg.  Dot, 
p.  140,  note.    Sachs:  Lehrb.,  1874,  p.  55.    Duchartre:  Elements  de  Bot., 
1877,  p.  102. 

2  Karsten  :  Gesammelte  Beitr.  zur  Anat.  u.  Phys.  d.  Pflanzen,  L,  1865, 
p.  253.    Dippel:  Das  Mikroskop,  I.,  p.  375.    Wiesner :  Technische  Mikro- 
skopie,  p.  83.    Weiss:  Allg.  Botanik.,  L,  p.  181 ;  Die  Pflanzenhaare,  Kar- 
stens  Bot.  Unters.,  I.     Nageli:  Das  Mikroskop,  1877,  P-  475- 

3  Cf.  Link  :  Grundlehrend.  Anat.,  1807,  p.  80,  where,  to  my  knowledge, 
it  is  mentioned  for  the  first  time  as  a  micro-chemical  reagent. 


34  MICRO-CHEMICAL   REAGENTS. 

CHLORATE  OF  POTASSIUM. 

This  reagent  is  used  with  nitric  acid,  either  in  a 
concentrated  aqueous  solution,  or,  better,  in  the 
crystalline  form,  for  the  destruction  of  the  "  middle 
lamella"  and  the  consequent  isolation  of  cells, 
especially  in  investigations  of  wood.1  The  mix- 
ture, the  so-called  Schultze  maceration-medium,  is 
boiled  for  a  few  minutes  in  contact  with  pieces  of 
the  tissue  to  be  studied.  After  careful  washing  in 
alcohol,  these  macerated  preparations  may  be  pre- 
served in  glycerine.  This  process  must  be  carried 
on  at  a  distance  from  microscopes  and  other  appar- 
atus that  can  be  injured  by  the  gases  developed. 

The  Schultze  mixture  has  also  been  found  useful 
as  a  reagent  for  suberin.  Thin  sections  are  boiled 
for  a  long  time  in  it ;  all  parts  of  the  cell-wall  soon 
become  clear,  but  those  which  contain  suberin 
possess  dark  and  sharp  contours  and  resist  the 
action  longer  than  the  others,  though  finally  they 
become  distorted,  abruptly  swell,  and  melt  to  form 
rounded  drops  of  eerie  acid,  which  are  soluble  in 
ether,  benzol,  chloroform,  caustic  potash,  and  boil- 
ing alcohol.  In  the  process  a  part  of  the  suberin 
is  dissolved  by  the  reagent,  only  a  part  being 
changed  into  eerie  acid.  Membranes  which  are 

l  Sanio:  Bot.  Zeitung,  1863,  p.  362,  note;  Anatomic  der  Kiefer.— 
Jahrb.  fur  wiss.  Bot.,  IX.  Hohnel :  Ueber  Kork.  —  Berichte  der  wiener 
Akad.,  1877,  Abth.  I.  Schacht :  Das  Mikroskop,  1855,  p.  27 ;  Anatom.  u. 
Physiol.  der  Gew.,  I.,  p.  14.  Dippel :  Das  Mikroskop  II.,  p.  101.  Nageli: 
Das  Mikroskop,  1877,  p.  474. 


INORGANIC     SALTS.  35 

only  slightly  suberiferous  will,  therefore,  hardly 
show  this  reaction  ;  and  to  detect  their  suberin, 
the  sections  are  placed  in  the  cold  fluid  a  few  mo- 
ments, and  then  removed  to  a  solution  of  potash. 
The  walls,  which  after  the  first  treatment  stand  out 
sharply,  assume  an  ochre-yellow  color  under  the 
action  of  the  potash  —  in  all  cases  after  being 
slightly  warmed. 


CUPRIC   SULPHATE  (Blue  Vitriol). 

This  substance  has  a  very  extensive  application 
in  micro-chemistry.1  It  is  always  used  in  a  tolera- 
bly concentrated  aqueous  solution,  and  must  be 
chemically  pure. 

It  is  used  in  the  following  (Trommer's)  test  for 
sugar.  A  moderately  thick  section  of  the  tissue 
which  is  to  be  studied  is  allowed  to  lie  from  two  to 
ten  minutes  in  a  concentrated  solution  of  the  salt. 
The  surface  is  then  rapidly  rinsed  with  distilled 
water,  and  the  section  transferred  to  a  boiling 
mixture  of  equal  parts  by  weight  of  water  and 
potassic  hydrate.  Cells  which  contain  cane  sugar 
(saccharose)  assume  a  bright  blue  color,  while  those 


_!  Nageli:  Das  Mikroskop,  1877,  pp.  475,  525.  Dippel :  Das  Mikro- 
skop,  I.,  p.  372 ;  II.,  p.  20.  Weiss  :  Allgem.  Botanik,  1878, 1.,  pp.  77, 171, 174. 
Sachs:  Flora,  1862,  p.  289;  Jahrbucher  fiir  wiss.  Bot.,  III.,  p.  187.  De 
Vries  :  Keimung  des  rothen  Klees.  —  Landwirths.  Jahrb.,  1877,  VI.,  p.  468. 
Wiesner :  Technische  Mikroskopie,  p.  79.  Fresenius :  Quantitative  chem- 
ische  Analyse,  Braunschweig,  1853,  p.  496.  Fehling:  Ann.  der  Chemie 
und  Pharmacie,  Bd.  LXXII.,  p.  106. 


36  MICRO-CHEMICAL    REAGENTS. 

which  contain  grape  sugar  (glucose}  become  clouded 
by  the  deposition  of  a  finely  granular  or  flocculent 
orange  precipitate  of  reduced  oxide  of  copper. 

By  this  test  we  are  thus  able  to  determine  which 
kind  of  sugar  was  present  in  the  tissue.  If  the 
blue  solution  of  cane  sugar  is  boiled  with  dilute 
sulphuric  or  nitric  acid  it  is  changed  into  grape 
sugar,  which  then  gives  the  red  reaction. 

Trommer's  test  also  serves  for  the  detection  of 
dextrine.  The  contents  of  cells  containing  this 
sugar  assume  a  vermilion  color,  while  small  gran- 
ules in  the  precipitate  exhibit  the  Brownian 
movement.  If  the  dextrine  is  mixed  with  pro- 
tein compounds,  however,  the  precipitate  is  yel- 
lowish. 

If  in  manipulation  too  much  of  the  cupric  sul- 
phate has  penetrated  the  cells  the  reaction  is  often 
masked  by  a  precipitate  of  cupric  hydrate.  To 
avoid  this  difficulty  Fehling's  fluid,  which  gives  the 
same  reaction,  may  be  used.  It  is  prepared  in  the 
following  manner.  Dissolve  4  gm.  of  cupric  sul- 
phate in  1 6  gm.  distilled  water;  and  16  gm.  of 
potassium  tartrate  in  the  minimum  of  water.  The 
two  solutions  are  to  be  mixed.  The  reagent 
should  be  kept  in  the  dark  and  needs  frequent 
renewal. 

[Prof.  W.  S.  Haines  has  found  in  glycerine  a 
very  desirable  substitute  for  the  tartrate  in  Feh- 
ling's test.  The  proportions  employed  by  him  for 
qualitative  examinations  are  :  Cupric  sulphate,  2 


INORGANIC    SALTS.  37 

gm. ;  potassic  hydrate,  6  gm.  ;  pure  glycerine,  7. 5 
cc. ;  distilled  water,  178  cc.1 — W.  T.] 

Arabin  (arabate  of  potassium)  Cerasin  (meta- 
gummate  of  potassium)  and  Bassorin  do  not  reduce 
the  Trommer  reagent ;  a  dark  blue  precipitate  only 
is  formed,  the  flocks  of  which  unite  into  balls  when 
heated. 

Protein  compounds  are  sometimes  recognized  by 
the  same  tests,  the  contents  of  young  cells 
assuming  a  beautiful  violet  color,  though  older  cells 
fail  to  show  the  reaction. 

Cell-walls  which  are  not  lignified  are  colored 
faintly  blue  by  long  soaking  in  an  aqueous  solution 
of  cupric  sulphate. 

POTASH  ALUM  (Alum).* 

An  aqueous  solution 3  is  employed  as  a  mordant, 
in  various  staining  processes,  e.  g.  in  Frey's  haem- 
atoxylin  and  Grenacher's  alum-carmine ;  as  an 
anhydrating  medium ;  or,  finally,  as  an  aid  in  the 
Hanstein  method  of  clearing  tissues,  q.  v. 


POTASSIC   NITRATE    (Niter,  Saltpeter). 
This  has  been  used  in  a  one-fourth  per  cent, 
aqueous  solution  as  a   culture-fluid  for  the  living 

1  Wheeler  :   Organic  Chemistry,  p.  187. 

2  The  ordinary  commercial  alum,  which  is  ammonium-alum,  is  quite  as 
useful. 

8  Frey  :  Das  Mikroskop,  p.  93.     Bachmann :   Dauerpra'parate,    p.    28. 
Hanstein:  Scheitelzellgruppe,  1868. 


OF  THE 
iiiuiWCQQITY 


38  MICRO-CHEMICAL  REAGENTS. 

tissues  of  higher  plants  during  observations  on  the 
division  of  the  nucleus.1 


MERCURIC  NITRATE  (Millon's  reagent).2 

This  is  made  by  dissolving  mercury  in  its  weight 
of  concentrated  nitric  acid,  and  diluting  with  an 
equal  volume  of  distilled  water.  It  should  be 
freshly  prepared  when  required  for  use. 

It  causes  the  cell-wall  to  swell,  and  so  renders 
its  striation  more  evident,  but  its  most  important 
use  is  for  the  detection  of  protein  compounds. 
These,  after  lying  in  it  for  some  time,  and  espe- 
cially after  slight  warming,  assume  a  very  red  color. 
The  surface  membrane  (Hautschicht)  of  proto- 
plasm is  slightly  colored  if  at  all.  It  should,  how- 
ever,, be  added  that  the  reagent  is  not  very  sensitive, 
and  the  reaction  is  not  always  obtained  (Nageli). 


CHLORIDE   OF  GOLD. 

A  one-half  per  cent,  solution  has  recently  been 
used  in  America  for  coloring  the  tissues  of  fungi. 
It  requires  from  one  to  six  hours  to  produce  the 

1  Treub:  Quelques  recherches  sur  le  role  du  noyau  dans  la  division  des 
cellules  vegetales,  1878,  p.  9.—  Naturk.  Verh.  d.  koningl.  Akad.,  Vol.  XIX., 
Amsterdam. 

2  Dippel :  Das  Mikroskop,  I.,  p.  281 ;  II.,  p.  18.  Nageli :  Das  Mikroskop, 
1877,  pp.  475,  527.    Weiss :  Allgem.  Bot.,  I.,  pp.  77,  144.     Millon:  Ann. 
de  Chim.  et  de  Phys.,  3  Ser.,  Tome  29,  p.  507.     Radlkofer :  Ueber  krystal. 
proteinart.  Kb'rper,  Leipzig,  1859. 


INORGANIC     SALTS.  39 

proper  effect.    Preparations  stained  with  it  may  be 
mounted  in  dilute  glycerine.1 

NITRO-PRUSSIATE  OF  SODIUM. 
This  is  useful  in  its  aqueous  solution  for  the 
detection  of  free  sulphur.2  It  should  be  prepared 
when  required  for  use.  The  crystals  need  to  be 
kept  from  the  air,  from  which  they  very  readily 
take  up  water.  The  preparation  to  be  tested  is 
heated  with  potassic  hydrate,  after  which  treat- 
ment the  granules  of  sulphur  unite  to  form  larger 
yellow  masses,  which  are  colored  violet  by  the 
nitro-prussiate. 

POTASSIC  FERRO-CYANIDE  (Yellow  Prussiate). 

An  aqueous  solution  precipitates  ferric  salts, 
thus  causing  a  blue  color.  This  reaction  has 
been  taken  advantage  of  for  the  detection  of  fer- 
ric hydrate  in  the  cell-wall,  e.  g.  in  Crenothrix.3 
Cells,  the  brown  color  of  whose  walls  causes  a 
suspicion  of  an  encrusting  compound  of  iron,  are 
treated  with  a  mixture  of  hydrochloric  acid  and 
the  ferro-cyanide.  If  the  beautiful  color  of  Prus- 
sian blue  appears  the  suspicion  caused  by  the 
brown  color  is  confirmed. 

1  W.  Hassloch:   New  York  Med.  Journ.,  Nov.  1878,  — also  Journal 
Royal  Microscop.  Soc.,  1879,  Vol.  II.,  p.  170.    I  have  not  tested  this  my- 
self. 

2  Cohn:    Untersuchungen    liber   Bacterien,  II.  —  Beitr.  zur  Biol.  d. 
Pfl.,  Bd.  I.,  Heft  3,  p.  175. 

8  Cohn;  Ueber  den  Brunnenfaden.  — -  Beitr.  zur  Biol.  d.  Pfl.,  Bd.  II., 
Heft  i,  p.  119. 


4O  MICRO-CHEMICAL  REAGENTS. 

SULPHO-CYANATE  OF  POTASSIUM. 

An  alcoholic  solution  is  employed,  sometimes  in 
combination  with  hydrochloric  acid,  for  the  detec- 
tion of  iron  in  the  cell-wall.  (See  the  second  sec- 
tion, Iron.) 

POTASSIUM  BICHROMATE. 

An  aqueous  solution  is  used  for  the  detection  of 
tannic  acid.1  Masses  of  tissue  of  considerable  size 
are  left  in  the  reagent  for  some  time.  Cells  which 
contain  tannin  assume  a  reddish-brown  color.  The 
iron-reactions,  however,  are  preferable. 

It  is  also  used  for  hardening  resin  masses. 

NITRATE   OF  SILVER. 

A  very  dilute  alkaline  solution  in  water  has  been 
employed  for  testing  the  living  condition  of  proto- 
plasmic bodies  which,  in  this  condition,  contain 
aldehyde.  According  to  Loew  and  Bokorny,2  the 
discoverers  of  this  reaction,  the  reagent  is  pre- 
pared as  follows  :  i.  A  one  per  cent,  solution  of 
nitrate  of  silver  is  made  in  distilled  water.  2.  A 
mixture  of  13  cc.  potassic  hydrate  solution  (s.  g. 
I-333)>  IO  cc-  ammonia  (s.  g.  .964),  and  77  cc.  dis- 

1  Dippel :   Das  Mikroskop,  I.,  p.  280.     Sanio  :  Bot.  Zeitung,  1863,  p. 
1 7.     Hanstein  :  Organe  der  Harz  und  Schleimabsonderung.  —  Bot.  Zeitung, 
1868,  p.  702.     Nageli:  Das  Mikroskop,  1877,  p.  475.     Weiss:  Allgem. 
Bot.,  I.,  p.  187,  note. 

2  Pflugers  Archiv.,  1881,  XXV. ;  Jahrb.  wiss.  Bot.,  1882. 


INORGANIC   SALTS.  4! 

tilled  water  is  made.  Before  using  these  solutions 
they  are  mixed,  i  cc.  of  each  being  taken,  and  the 
mixture  diluted  so  as  to  make  a  liter.  The  mixture 
can  be  made  only  at  the  moment  when  it  is  to  be 
employed ;  otherwise  metallic  silver  is  precipitated 
by  the  light. 

This  reagent  colors  living  protoplasm  black, 
while  dead  protoplasm  remains  uncolored.  The 
reaction  may  be  obtained  with  a  solution  diluted 
very  greatly  (even  i  :  i, 000,000).' 

Tannic  acid  also  gives  a  reaction,  but  this 
is  effected  only  with  a  less  dilute  solution 
(i  :  10,000). 

Glucose  gives  a  reaction  with  a  solution  of 
i  :  100,000,  the  cells  coloring  brown,  and  not  black. 
In  this  proportion,  therefore,  nitrate  of  silver  is  an 
excellent  reagent  for  this  sugar. 

1  These  very  dilute  solutions  cannot  be  employed  in  as  small  quantity 
as  micro-chemical  reagents  usually  are.  To  obtain  good  results  it  is  neces- 
sary, according  to  Bokorny,  to  immerse  a  few  cells  (e.  g.  of  Spirogyra)  in  a 
large  quantity  (.5  —  i  liter)  of  the  reagent  for  six  to  twelve  hours. — 
French  Translator. 


42  MICRO-CHEMICAL  REAGENTS. 


ORGANIC   SALTS. 


ACETATE  OF  IRON 

Is  used  in  an  aqueous  solution,  as  has  been  de- 
scribed for  the  chloride. 


ACETATE  OF  COPPER  (Verdigris) 

Is  used  as  a  means  of  recognizing  resins.  Masses 
of  tissue  are  soaked  five  or  six  days  in  an  aqueous 
solution  of  the  salt,  after  which  treatment  the 
resinous  masses  assume  an  emerald  green  color.1 


SULPHATE  OF  ANILIN. 

An  aqueous  solution  of  this  substance,  the  so- 
called  Wiesner  anilin  reagent,  is  used  for  the  detec- 
tion of  lignin,  the  constituent  of  wood.2  The 
sections  are  first  placed  in  a  dilute  solution  of  the 
sulphate  until  they  are  well  saturated  with  it ;  even 
with  no  other  treatment,  lignified  membranes  often 
assume  a  faint  yellow  color,  which,  however,  is 

1  Franchimont :   Origine  et  constitution  chimique  des  r6sines  de  ter- 
penes.—  Cf.  Archives  Neerlandaises,  1871,  T.  VI.,  p.  427,  with  PI.  8. 

2  Wiesner:  Technische  Mikroskopie,  1867^.64,     Karsten :  Bot.  Un- 
tersuchungen,   I.,   p.    120,   note.      B urger stein :    Sitzungsber.   der  wiener 
Akad.,  1874,  Bd.  LXX.,  Abth.  r,  p.  338.     Hohnel :   Ueber  Kork,  etc.— 
Sitzungsber.  der  wiener  Akad.,  1877,  Bd.  LXXVL,  Abth.  i,  p.  21.     - 


ORGANIC   SALTS.  43 

intensified  by  transferring  the  section  to  dilute 
sulphuric  acid.  If  it  is  desired,  the  reagents  may 
be  mixed  before  using. 

Since  this  anilin  salt  is  found  in  the  market  only 
in  a  very  impure  and  barely  soluble  form,  it  is  bet- 
ter to  replace  it  by  the  next. 

CHLORIDE  OF  ANILIN 

Is  used  in  aqueous  solution  for  the  same  purposes 
as  sulphate  of  anilin,  and  in  the  same  manner, 
except  that  the  acid  used  must  be  hydrochloric.1 
An  alcoholic  tincture  of  either  anilin  salt  may 
replace  the  aqueous  solution,  and  the  colors  pro- 
duced are  then  more  intense. 

CHLORAL. 

This  has  been  recently  introduced  into  micro- 
chemistry  by  Meyer.2  It  is  employed  in  aqueous 
solution :  five  parts  of  chloral  to  two  parts  of 
water;  and  should  be  used  at  a  temperature  of  15° 
C,  as  crystals  are  precipitated  at  lower  tempera- 
tures. Its  effect  upon  fats  and  volatile  oils  is 
similar  to  that  of  alcohol.  It  dissolves  the  same 
saccharine  and  amylaceous  matters  (Kohlenhy- 
drate)  as  water,  and  causes  the  swelling  of  starch 
grains.  It  swells  or  dissolves  protein  matters,  and 
is,  therefore,  frequently  useful  in  clearing  tissues. 

1  Hohnel:  Ueber  Kork,  etc.,  p.  21. 

2  Arthur  Meyer :  Das  Chlorophyllkorn,  Leipzig,  1883. 


/]/)         MICRO-CHEMICAL  REAGENTS. 

OTHER  ORGANIC  COMPOUNDS. 


SOLUTION  OF  CANE  SUGAR  (Syrup). 

Vegetable  cells  containing  much  sugar  often 
assume  a  beautiful  red  color  on  the  addition  of 
sulphuric  acid.  This  fact  has  been  utilized  in  ob- 
taining a  reaction  for  protoplasmic  substances. 
The  tissues  to  be  tested  are  first  saturated  with  a 
solution  of  cane  sugar  and  water.  On  the  addition 
of  sulphuric  acid  the  red  color  manifests  itself. 
This  reaction  (Raspail's)1  is,  however,  often  difficult 
to  obtain,  and  not  very  good.  The  sections  have 
to  be  left  in  the  acid  for  some  time  before  the  color 
appears. 

Syrups  of  different  degrees  of  concentration  also 
find  application  as  anhydrating  media.2  A  three 
per  cent,  solution  had  been  used  as  a  fluid  of  prep- 
aration in  the  study  of  transparent  ovules,  e.  g. 
embryo  sac  of  Monotropa  and  Orchis ;  and  a  five 
per  cent,  solution  has  been  recommended  for 
pollen-cultures  under  the  microscope. 

1  Raspail :  Chimie  organique,  1839,  II.,  p.  139.  M.  Schultze  :  Ann.  der 
Chem.  u.  Pharm.,  Bd.  LXXL,  p.  270.  (Second  discovery  of  the  reaction.) 
Schacht :  Mikroskop,  p.  27 ;  Pflanzenzelle,  pp.  27,  28 ;  Anat.  u.  Physiol. 
der  Gewachse,  I.,  pp.  46,  61.  Dippel :  Mikroskop,  I.,  p.  283.  Nageli : 
Mikroskop,  1877,  pp.  476,  526.  Frey :  Mikroskop,  p.  73.  Hofmeister: 
Pflanzenzelle.  —  Handb.  der  phys.  Bot,  I.,  p.  2.  Weiss  :  Allg.  Bot.,  I.,  p. 
77.  Duchartre:  Elements  de  Bot,  2  ed.,  p.  25. 
2  Strasburger:  Befruchtung  u.  Zelltheilung,  pp.  16,  29,  52,  etc. 


OTHER  ORGANIC   COMPOUNDS.  45 


ASPARAGIN. 

Borodin  x  has  recommended  a  tepid  concentrated 
aqueous  solution  for  the  detection  of  asparagin, 
which  has  been  precipitated  in  the  tissue  by  alcohol 
(p.  27),  on  the  principle  that  a  crystallized  sub- 
stance is  insoluble  in  a  saturated  solution  of  the 
same  substance. 

Asparagin  is  prepared  by  evaporating  the  well- 
boiled  and  filtered  sap  of  young  seedlings  of  legu- 
minosae  (especially  lupines)  which  have  germinated 
in  the  dark,  or  by  the  evaporation  of  the  dialyzed 
aqueous  decoction  of  althaea  root ;  when  it  sepa- 
rates as  crystals. 


DIPHENYLAMIN. 

This  officinal  substance  has  of  late  been  used  as 
a  reagent  for  nitrates  and  nitrites.2  The  sections 
to  be  tested  are  allowed  to  dry  on  the  slide  and 
afterwards  moistened  with  a  solution  of  .01-.  I  gm. 
of  diphenylamin  in  10  cc.  pure  sulphuric  acid. 
Even  very  small  quantities  of  compounds  contain- 
ing nitric  acid  are  indicated  by  the  appearance  of 
a  dark  blue  color  in  the  cells ;  the  reaction  is  very 
distinct. 


1  Borodin :  Bot.   Zeitung,  1878,   p.   804,  et  seq.     Detmer :  Physiol.  u. 
Keimung  d.  Sam  en,  1880,  p.  171,  et  seq. 

2  Molisch :  Ueber  den  Mikrochemischen   Nachweis  von  Nitraten  und 
Nitriten  etc.  — Ber.  d.  deutschen  hot.  Ges.,  1883, 1.,  p.  15*. 


46  MICRO-CHEMICAL   REAGENTS. 

BRUCIN 

Has  been  introduced  into  micro-chemistry  for  the 
same  purpose  as  diphenylamin,  but  it  does  not 
serve  so  well  for  the  detection  of  small  quantities 
of  nitrates  and  nitrites.  Molisch  x  recommends  a 
solution  of  .2  gm.  brucin  in  10  cc.  pure  sulphuric 
acid.  A  dry  section  of  a  tissue  which  contains 
N2  O5  assumes  a  bright  red  or  reddish-yellow  tint 
when  placed  in  a  drop  of  the  solution. 

INDOL. 

This  substance  has  been  employed  quite  recently 
by  Niggl  as  a  reagent  for  lignin,  or  lignified  mem- 
branes.2 The  discovery  of  this  reaction  is  due  to 
Professor  Baeyer,  of  Munich.3  Several  crystals  of 
indol  are  dissolved  in  a  sufficient  quantity  of  warm 
distilled  water.  The  sections  to  be  tested  are 
placed  in  a  drop  of  the  reagent  for  several  minutes, 
after  which  they  are  washed  in  dilute  sulphuric 
acid  (i  pt.  :  4  pts.  water).  Lignified  cell-walls 
assume  a  very  intense  red  color. 

PHLOROGLUCIN. 

One  of  the  prettiest  and  best  reactions  of  micro- 
chemistry  has  recently  been  discovered  by  Wiesner ; 

1  Molisch  :  Ber.  deutsch.  bot.  Ges.,  1883,  I.,  p.  150. 

2  Niggl:  Flora,  1881,  No.  35,  p.  545. 

3  Ann.  d.  Chem.  u.  Pharm.,  Bd.  CXL. 


OTHER  ORGANIC  COMPOUNDS.         4/ 

viz.  the  phloroglucin  test,  for  lignin.  An  aqueous 
solution  or,  better,  an  alcoholic  tincture  of  the 
reagent  is  employed,  and  even  in  excessively  small 
quantity  induces  the  reaction.1  The  section  to  be 
studied  is  treated  with  hydrochloric  acid,  and  then 
placed  in  a  drop  of  phloroglucin  on  the  slide.  The 
parts  containing  lignin  assume  a  beautiful  and  in- 
tense rose-red  color  with  a  rapidity  depending  upon 
the  concentration  of  the  solution.  The  prepara- 
tions may  be  kept  for  a  considerable  time.  If 
difficulty  is  experienced  in  getting  the  reagent  — and 
at  present  it  is  quite  expensive  and  hard  to  obtain  — 
an  extract  of  cherry  wood  diluted  with  water  may 
replace  it.  This  contains  the  substance  in  question 
among  others,  but  gives  a  more  violet  reaction  than 
the  pure  reagent.2 


ROSOLIC   ACID 

Is  recommended  by  Janczewski 3  as  "  the  best  of  the 
reagents  which  color  the  callosities  of  sieve  tubes." 
A  little  ammonia  or  sodic  carbonate  should  be 
added  to  it. 

1  Wiesner:  Sitzungsber.   der  wiener  Akad,  Bd.  LXXVIL,  Abth.  i., 
Januarheft. 

2  This  has  been  called  the  xylofilin  reaction  by  its  discoverer,  Hohnel.  — 
Sitzungsanzeiger  d.  wiener  Akad.,  1877,  No.  23,  pp.  228,  229. 

3  Etudes  comparees  sur  les  tubes  cribreux.  —  Mem.  de  la  Soc.  das  Sc. 
nat.   de    Cherbourg,   1881,  XXIII.,  p.   350.      The    discovery   is   due  to 
Szyszyloviez. 


48  MICRO-CHEMICAL    REAGENTS. 


COLORING  AGENTS. 


Lately  various  dyestuffs  have  been  employed  for 
the  differentiation  of  the  tissue  systems,  as  well  as 
for  the  recognition  of  some  of  the  cell  contents ; 
of  these  only  the  most  important  will  be  mentioned. 


TINCTURE  OF  ALCANNA. 

A  red  dye,  extracted  by  alcohol  from  the  root  of 
Alcanna  tinctoria.  is  employed  for  coloring  resins, 
which  have  a  special  avidity  for  this  substance. 
Protoplasm  is  also  colored  pale  red  by  it.1  Prepa- 
rations colored  by  alcanna  do  not  endure  drying. 

COCHINEAL. 

An  aqueous  extract  of  cochineal  is  employed 
with  acetic  acid  or  alum,  for  staining  the  prosen- 
chyma  (bast  cells)  of  the  phloem  in  fibro-vascular 
bundles.  After  lying  in  the  dye  for  some  time, 
these  cells  assume  an  intense  red  color,  while  the 
other  elements  are  not  affected,  or  but  slightly 
colored.  Yet  there  are  certain  kinds  of  wood 
which  imbibe  the  cochineal  extract,  but  after-treat- 

1  N.  J.  C.  Miiller :  Untersuchungen  liber  die  Vertheilung  der  Harze.  — 
Jahrbiicher  fiir  wiss.  Bot.,  V.,  p.  387.  Hanstein:  Organe  der  Harz-  und 
Schleimabsonderung.  —  Bot.  Zeitung,  1869,  pp.  707,  708. 


COLORING    AGENTS.  49 

ment  with  hydrochloric  or  sulphuric  acid  removes 
the  color  from  everything  except  the  bast  cells,  in 
which  it  becomes  more  intense. 

As  a  coloring  agent  in  the  investigation  of  pro- 
tein bodies,  this  substance  has  also  found  use.1 

CARMINE. 

A  solution  of  carmine  in  dilute  potash,  such  as 
is  sold  by  dealers,  is  used  for  staining  the  nuclei  of 
cells.2  The  solution,  which  should  contain  very 
little  undissolved  carmine,  is  filtered  and  then 
mixed  with  alcohol  or  glycerine  in  various  propor- 
tions. The  object  requires  to  lie  in  the  solution 
for  some  time.  Only  the  nucleus  (and  protein 
grains)  imbibe  the  color. 

The  carminate  of  ammonium  (ammonia  carmine) 
is,  however,  more  commonly  used.3  It  is  prepared 
in  the  following  manner,  suggested  by  Hartig. 
Carmine  powder  is  dissolved  in  a  strong  solution 
of  ammonia  until  this  is  saturated  ;  the  solution 
is  evaporated  to  dryness  over  a  water  bath,  and 
the  carminate  thus  formed  is  prepared  for  use  by 
solution  in  water. 

1  Wigand:  Bot.  Zeitung,  1862,  pp.  129,  139.     Maschke:  Bot.  Zeitung, 
1859,  p.  22.  Vogl ;    Anat.  u.  Histol.  der  unterirdischen  Theile  von  Convol- 
vulus arvensis.  —  Sitzungsber.  d.  wiener  Akad.,  1863,  XIII. 

2  Hartig:  Der  Fiillkern,  etc.  —  Karsten's  botan.  Untersuchungen ,    I., 
p.  282,  note.. 

3  Dippel :  Das  Mikroskop,  I.,  p.  184.  Frey:  Mikroskop,  1873,  PP-  ^7?  88, 
90.     Bachmann :  Dauerpraparate,  p.  26.     Tangl :  Protoplasma  der  Erbse. 
—  Sitzungsber.  der  wiener  Akad.,  Bd.  XXVI.-XXVIII. 


5<D  MICRO-CHEMICAL   REAGENTS. 

Thiersch  gives  the  following  formula  for  its  pre- 
paration. One  part  of  carmine  is  dissolved  in  one 
part  (by  weight)  of  concentrated  ammonia  and 
three  parts  of  distilled  water.  This  solution  is 
mixed  with  eight  times  its  volume  of  dilute  oxalic 
acid  (i  :  22).  Twelve  volumes  of  absolute  alcohol 
are  then  added,  and  the  whole  is  filtered.  The 
filtrate  may  be  rendered  more  orange  in  color  by 
the  addition  of  oxalic  acid,  or  more  violet  by  the 
addition  of  ammonia.  If  oxalate  of  ammonium  is 
precipitated,  it  can  be  removed  by  filtration  or  redis- 
solved  by  the  addition  of  a  few  drops  of  ammonia. 

Grenacher's  alum-carmine,  recently  introduced 
into  vegetable  histology  by  Tangl,1  is  prepared  by 
him  in  the  following  manner.  A  saturated  aqueous 
solution  of  alum  is  made.  The  desired  quantity 
of  carmine  is  dissolved  in  it,  the  solution  is  boiled 
for  about  ten  minutes,  allowed  to  cool,  and  filtered. 
Walls  consisting  of  cellulose  are  colored  bright-red 
by  this  preparation,  while  those  containing  su- 
berin  or  lignin  remain  unstained.  Protoplasmic 
bodies  and  the  nuclei  of  cells  are  also  stained  with 
difficulty,  and  but  slightly.  It  is  recommended 
that  parts  of  plants  from  which  sections  are  to  be 
cut,  shall  be  hardened  in  absolute  alcohol,  as  this 
increases  the  power  of  the  membranes  to  take  up 
the  coloring  matter. 

1  Tangl .  Ueber  offene  Communication  zwischen  den  Zellen  des  Endo- 
sperms.—  Jahrb.fiir  wiss.  Bot,  1880,  XII.,  p.  170.  Grenacher  :  Arcliiv.  f. 
mikr.  Anatomic,  1879,  P-  4^5  *>  Zeitschrift  f.  Mikroskopie,  1879, 
II.,  p.  55- 


COLORING   AGENTS.  51 

If  the  preparation  should  be  too  deeply  stained 
by  this  carmine  fluid,  it  can  be  bleached  in  an 
alcoholic  solution  of  oxalic  acid. 

Carmine  stains  all  protoplasm  if  allowed  to  act 
a  sufficient  length  of  time.  The  nuclei  of  cells  are 
most  deeply  colored.  Living  protoplasm  does  not 
imbibe  the  coloring  matter.  This  occurs  only 
after  it  has  been  killed  by  the  addition  of  the 
reagent.  In  general  it  may  be  said  that  it  colors 
most  vegetable  albuminoids,  while  starch  and 
cellulose  take  it  up  in  much  smaller  quantity  or 
not  at  all. 

[The  double-staining  of  clear  sections,  usually 
bleached  by  the  action  of  Labarraque's  solution,  or 
some  similar  fluid,  is  capable  of  yielding  very 
good  results  where  convenience  of  demonstration 
in  the  class-room  is  concerned.  As  good  direc- 
tions as  any  are  those  of  Dr.  Rothrock,1  who  uses 
Woodward's  ammonia  carmine  and  the  aniline 
color  known  as  iodine-green. 

The  section  is  placed  in  alcohol  faintly  colored 
by  the  addition  of  a  few  drops  of  a  concentrated 
tincture  of  the  green,  where  it  is  allowed  to  remain 
from  twelve  to  twenty-four  hours,  according  to 
circumstances.  It  is  then  successively  passed 
through  a  series  of  fluids  in  the  following  order : 
one,  water ;  two,  carmine  ;  three  to  five,  alcohol ;  six, 
absolute  alcohol ;  seven,  oil  of  cloves  :  being  merely 

1  Rothrock :  Staining  and  double-staining  of  vegetable  tissues.  —  Bot. 
Gazette,  Sept.  1879,  Vol.  IV.,  pp.  201-6. 


5  2  MICRO-CHEMICAL  REAGENTS. 

dipped  into  the  water,  remaining  from  twenty  sec- 
onds to  a  minute  in  the  carmine,  being  well  rinsed 
in  the  first  watch-glass  of  alcohol,  and  staying  ten 
to  twenty  minutes  in  each  of  the  others.  It  is 
left  in  the  oil  of  cloves  until  cleared  up,  when 
it  is  ready  for  mounting  in  balsam.  The  gen- 
eral experience  of  teachers  seems  to  be,  how- 
ever, that  more  time  is  consumed  in  making  the 
few  successful  preparations  than  they  are  worth. 
—  W.  T.] 

Beale's  carmine,  which  is  especially  useful  in  dif- 
ferentiating the  nucleus,  is  prepared  by  dissolving 
.6  gm.  carmine  in  2  gm.  boiling  ammonia  water. 
The  solution  is  set  aside  for  an  hour  to  allow 
some  of  the  ammonia  to  escape.  Sixty  gm.  dis- 
tilled water,  60  gm.  glycerine,  and  15  gm.  absolute 
alcohol  are  then  added.  After  standing  for  some 
time,  the  fluid  is  filtered  and  ready  for  use.1 

Strasburger,  in  the  study  of  the  embryo  sac, 
stains  the  protoplasm  with  a  boracic  solution  of 
carmine,  prepared  as  follows  :  Four  parts  borax 
are  dissolved  in  fifty-six  parts  distilled  water.  To 
this  one  part  of  carmine  is  added.  One  volume 
of  this  solution  is  diluted  with  two  volumes  abso- 
lute alcohol  and  filtered.  By  the  use  of  this  dye 
the  study  of  the  forms  of  the  nucleus  is  greatly 
facilitated.  The  preparations  may  be  preserved 
in  glycerine  or  glycerine  jelly.2 

1  Frey :  Mikroskop,  p.  90. 

2  Strasburger:  Zellbild.  u.  Zelltheilg.,  1880,  p.  9. 


COLORING   AGENTS.  53 

Czokor  recommends  the  following  coloring  fluid : 
7  gm.  cochineal  are  pulverized  with  an  equal  quan- 
tity of  calcined  alum.  This  is  dissolved  in  700 
e:m.  distilled  water,  and  the  whole  is  boiled  down 
to  about  400  gm.  After  it  is  cool  a  drop  of  car- 
bolic acid  is  added,  and  the  fluid  is  filtered.  It 
should  last  at  least  six  months  without  deteriora- 
tion. At  the  end  of  this  time  it  should  be  re- 
filtered  after  the  addition  of  another  drop  of  car- 
bolic acid.1 


PICROCARMINATE   OF  AMMONIUM  (Picrocarmine). 

This  staining  agent,  which  is  much  used  by 
students  of  animal  histology,  is  employed  in  botani- 
cal micro-chemistry,  chiefly  for  differentiating  the 
nucleus.2  It  is  prepared  by  adding  a  strong  solu- 
tion of  ammonium-carminate  to  a  concentrated 
aqueous  solution  of  picric  acid,  until  this  is  neu- 
tralized. After  evaporating  it  to  four-fifths  its 
original  volume,  it  is  set  aside  for  a  time,  and  then 
filtered,  when  the  dark  orange  fluid  is  ready  for 
use.3  Another  method  has  been  recommended  by 
Gage.  Equal  parts  by  weight  of  picric  acid  and 

1  Czokor:   Die  Cochenille  Carminlosung. —  Arch.  f.  mikr.  Anat.,  1880, 
XVIIL,  p.  412  et  seq.  —  See  also  Bot.  Centralblatt,  1880,  p.  1280. 

2  Treub:  Actes  du  congres  international  a  Amsterdam,  1877,  Leyden, 

1879,  P-  J46. 

8  Frey:  Mikroskop,  p.  91.  Bachmann:  Dauerpraparate,  p.  27. 
Treub:  Role  du  noyau  dans  la  division  des  cell.,  1878,  p.  23.  Pelletan : 
Le  Microscope,  1870,  p.  207.  Gage:  American  Monthly  Micr.  Journ., 

1880,  p.  22;  Journal  of  the  Royal  Micr.  Soc.,  1880,  Vol.  III.,  p.  501. 


UNIVERSITY 


54  MICRO-CHEMICAL  REAGENTS. 

carmine  are  dissolved  ;  the  former  in  one  hundred 
parts  of  water,  the  latter  in  fifty  parts  of  concen- 
trated ammonia.  The  solutions  are  then  mixed 
and  filtered,  evaporated  to  dryness,  and  the  residue 
is  redissolved  in  a  hundred  times  its  weight  of 
water. 

Protoplasm  is  colored  a  yellowish-red  by  it.  The 
nucleus  quickly  assumes  a  deeper  color,  especially 
after  very  short  action  of  the  coloring  matter.  The 
best  degree  of  concentration  is  a  one  per  cent, 
solution.  Maupas  recommends  the  use  of  alcohol, 
picro-carmine,  and  glacial  acetic  acid  for  staining 
the  nucleus.1 

[Mayer's  picro-carmine2  is  prepared  as  follows  :  — 
"To  a  mixture  of  powdered  carmine  (2  g.)  with 
water  (25  cc.),  while  heating  over  a  water-bath, 
add  sufficient  ammonia  to  dissolve  the  carmine. 
The  solution  may  then  be  left  open  for  a  few 
weeks  in  order  that  the  ammonia  may  evaporate ; 
or  the  evaporation  may  be  accelerated  by  heating 
(Hoyer).  So  long  as  any  ammonia  remains  large 
bubbles  will  form  while  boiling,  but  as  soon  as  the 
free  ammonia  has  been  expelled  the  bubbles  will 
be  small,  and  the  color  of  the  fluid  begin  to  be  a 
little  lighter.  It  is  then  allowed  to  cool,  and  fil- 
tered. To  the  filtered  solution  is  added  a  concen- 
trated aqueous  solution  of  picric  acid  (about  four 

1  Maupas :  Comptes  rendus,  July  1879,  No.  4,  p.  250. 

2  Mayer:    M  T.  Zool.  Stat.  Neapel,  1880,  II.,  pp.  1-27.  — Journ.  Roy. 
Mic.  Soc.,  Dec.  1882,  Ser.  2,  Vol.  II.,  pp.  876-7. 


COLORING     AGENTS.  55 

volumes  of  the  acid  to  one  of  the  carmine  solu- 
tion). The  addition  of  the  acid  should  cease  before 
a  precipitate  begins  to  form.  In  order  to  protect 
this  fluid  against  changes  attributed  to  bacteria  by 
Hoyer,1  Dr.  Mayer  places  a  small  crystal  of  thymol 
in  the  containing  bottle ;  Hoyer  uses  chloral-hydrate 
(i  per  cent,  or  more)  for  the  same  purpose." 

Weigert 2  prepares  the  reagent  by  the  following 
process  :  "  Over  2  gm.  of  carmine  are  poured  4  gm. 
common  ammonia,  and  the  whole  left  twenty-four 
hours  in  a  place  protected  against  evaporation  ; 
200  gm.  of  a  concentrated  picric  acid  solution  are 
then  poured  in  ;  the  mixture  is  left  twenty-four 
hours,  until  all  soluble  matters  are  dissolved.  Very 
small  quantities  of  acetic  acid  are  then  added,  until 
a  slight  precipitate  comes  down  even  after  stirring ; 
a  rather  copious  precipitate  is  usually  thrown  down 
in  the  course  of  the  next  twenty-four  hours;  it 
should  be  removed  by  filtration.  A  picro-carmine 
which  does  not  stain  readily  may  be  improved  by 
the  addition  of  acetic  acid." — W.  T.] 

H^MATOXYLIN.3 

This  substance  is  the  active  principle  in  the  ex- 
tract of  logwood,  but  is  not  found  in  great  quantity 
in  the  tinctura  ligni  campeschiani.  It  may  be 

1  Hoyer:  Beitr.  z.  histol.  Technik.  —  Biol.  Centralblatt,  1882,  II.,  pp. 
17-19. 

2  Arch,   pathol.  Anat.  (Virchow),  1881,  Vol.  84,  pp.  275-294.  — Journ. 
Roy.  Micr.  Soc.,  Feb.  1883,  Ser.  2,  Vol.  III.,  p.  139. 

3  Frey :  Mikroskop,  p.  91.  Pelletan :  Le  Microscope,  p.  209.    Ranvier  : 


56  MICRO-CHEMICAL   REAGENTS. 

obtained  in  the  market  ready  prepared.  The  stain- 
ing fluid  is  made  by  dissolving  .35  gm.  haematoxy- 
lin  in  10  gm.  water.  To  this  are  added  a  few  drops 
of  an  alum  solution  (which  acts  as  a  mordant  in 
fixing  the  color),  made  by  dissolving  3  gm.  alum  in 
30  gm.  water.  This  makes  a  beautiful  violet  fluid, 
which  colors  the  nucleus  deep  blue.  It  is  the  best 
staining  fluid  that  is  now  known  for  the  nucleus.1 
Preparations  need  to  lie  in  it  for  some  time.  They 
may  be  preserved  in  glycerine. 

I  have  used  with  success  the  method  of  staining 
bacteria  first  published  by  Koch.  The  dried 
preparation  is  treated  with  a  concentrated  extract 
of  Campeachy-wood  in  water.  After  removing  the 
superfluous  dye  with  distilled  water,  the  color  is 
fixed  by  the  use  of  dilute  chromic-acid.  The  prep- 
aration can  be  preserved,  after  drying,  in  glycerine 
or  Canada  balsam.  Cilia  and  the  bodies  of  the 
cells  are  sharply  differentiated  by  this  method.2 
Koch  has  later  recommended  coloring  with  haem- 
atoxylin.  Staff-shaped  bacteria,  however,  do  not 
color  by  this  substance,  according  to  him.3  I  have 
however  used  the  haematoxylin  tincture  with  suc- 
cess even  on  certain  staff-shaped  bacteria.  After 
rinsing,  my  preparations  are  preserved  dry. 

Histologie,  p.  103.  Robin:  Microscope,  1877,  p.  250.  Bachmann:  Dauer- 
praparate,  p.  28.  Schmitz :  Niederrhein.  Gesellsch.,  Nov.  1879. 

1  Johow:  Zellkerne  d.  hoheren  Monocotylen.,  Diss.,  Bonn,  1880,  p.  9, 
note. 

2  Koch :  Conserviren  und  Photographiren  der  Bacterien. —  Cohn's  Beitr. 
z.  Biol.  d.  Pfl.,  II.,  p.  421. 

3  Wundinfectionskrankheiten,  1878,  p.  30. 


COLORING    AGENTS.  57 


NIGRO&N. 

[Errera  recommends x  this  tar  derivative  for 
staining  the  nucleus.  It  is  soluble  in  water,  but 
insoluble  in  alcohol  and  ether.  After  remaining  a 
short  time  in  a  solution  of  nigrosin,  the  section  is 
transferred  to  water,  where  it  remains  until  no 
more  of  the  color  is  removed,  when  it  may  be 
mounted  in  glycerine  or  glycerine  jelly,  or  trans- 
ferred to  alcohol,  and  afterward  cleared  in  oil  of 
cloves  and  mounted  in  balsam.  The  latter  me- 
dium serves  best  for  specimens  intended  to  show 
the  chromatin ;  while  the  former  are  preferable 
for  those  intended  to  show  the  achromatin  of 
Flemming.  —  W.  T.] 

EOSIN. 

This  beautiful  rose-colored  derivative  of  phthalic 
acid  has  a  pronounced  green  fluorescence.  It  is 
employed  in  aqueous  [or  alcoholic]  solution. 
Even  a  very  small  quantity  has  great  coloring 
capacity.2  It  has  been  used  for  staining  Sarcina 
and  Sarcinoglobulus.  It  does  not  appear  adapted 
for  use  on  bacteria  (Bacillus,  Bacterium,  etc.).  [I 
have  succeeded  in  obtaining  fair  preparations  of 
Bacillus  subtilis  by  staining  in  alcohol-eosin,  and 

1  Proces-verbal  de  la  seance  mensuelle  du  25  Juin,  1881,  Soc.  beige  de 
Microscopic,  p.  CXXXIV. 

"  Poulsen :  Om  nogle  mikroskopiske  Planteorganismer.  —  Nat.  Foren. 
vidsk.  Meddel.,  1879-80,  p.  235.  Preparations  stained  with  eosin  may  be 
preserved  in  glycerine. 


5  8  MICRO-CHEMICAL   REAGENTS. 

mounting  in  balsam.  —  W.  T.]  In  the  tissues  of 
higher  plants,  where  its  effect  has  not  yet  been 
fully  studied,  it  stains  dead  protoplasm  a  beautiful 
rose  color.  Eosin  is  especially  useful  for  coloring 
the  plasma  of  sieve  tubes  and  the  nuclei  of  cells. 
It  has  also  been  used  as  the  first  dye  in  double- 
staining  preparations,  which  are  afterwards  treated 
with  Nicholson's  blue,  fixed  with  absolute  alcohol 
and  mounted  in  dammar.1 


ANILINE   COLORS.2 

Within  a  few  years  the  beautiful  aniline  colors 
have  been  generally  applied  in  many  histological 
researches.  In  none,  however,  are  they  more 
useful  than  in  the  preparation  of  bacteria,  not  only 
in  sections  of  animal  tissues,  but  also  in  films  ob- 
tained by  drying  (and  hardening  through  a  flame) 
a  thin  layer  of  fluid  containing  these  organisms 
on  the  cover-glass.  The  discovery,  by  Weigert, 
in  1871,  of  the  power  of  carmine  to  color  bacteria  3 
led  him  to  try  the  effect  of  the  aniline  salts,  in 
which  a  very  energetic  coloring  matter  —  almost 
a  reagent  for  bacteria  —  was  found.  The  names 

1  Journ.  of  Roy.  Micr.  Soc.,  1880,  III.,  p.  693. 

2  VVigand:  Bot.  Zeitung,  1862,  p.  17.  Hanstein  :   Organe  der  Harz-  und 
Schleimabsonderung  in  den  Laubknospen.  —  Bot.  Zeitung,  1868,  p.  708. 

3  The  "  sulphate  of  rosanilin  "  has  been  strongly  recommended  by  Salo- 
monsen  for  staining  bacteria  in  putrid  blood.     It  is  used  in  a  concentrated 
aqueous  solution  prepared  by  heating,  and  filtered  after  it  has  become  cold. 
—  Cf.  Studier  over  Blodets  Forraadnelse,  Copenhagen,  1877,  p.  15. 


COLORING   AGENTS.  59 

of  Ehrlich  and  Koch  are  also  well-known  in  con- 
nection with  the  further  improvement  of  this 
method  of  research. 

In  the  following  paragraphs  only  some  of  the 
most  useful  aniline  dyes  will  be  mentioned ;  their 
number  is  daily  increasing.  In  general  it  may  be 
stated,  that  all  preparations  stained  by  aniline 
colors  should  be  carefully  washed  before  being 
mounted;  they  must  also  be  kept  in  the  dark,  as 
most  of  these  colors  soon  fade  in  the  light. 


FUCHSIN, 

In  an  alcoholic  tincture,  colors  especially  well  thick- 
ened cell-walls  ;  the  different  layers  often  with  dif- 
ferent intensity.  Sections  which  are  to  be  stained 
should  be  free  from  potash,  which  destroys  the 
color,  and  they  must  be  treated  in  alcohol,  the  addi- 
tion of  water  precipitating  the  dye.  Stained  prepa- 
rations remain  unchanged  only  for  a  limited  time. 
Fuchsin  has  recently  been  applied  by  Ehrlich x 
similarly  to  vesuvin,  with  methylene  blue,  for  the 
staining  of  the  Bacillus  of  tuberculosis. 

HANSTEIN'S  ANILINE  VIOLET 

Is  prepared  by  mixing  about  equal  parts  of  methyl 
violet  and  fuchsin,  and  dissolving  them  in  alcohol. 
Its  action  depends  upon  the  different  avidity  of 

1  Ehrlich:  Zeitschr.  f.  klin.  Medicin,  1882,  II. 


6O  MICRO-CHEMICAL   REAGENTS. 

the  various   substances  which   are  found   in   the 
tissues  or  cells  for  the  mixed  colors. 

Protoplasm  is  stained  violet-blue.  Amyloid  sub- 
stances, the  nucleus  and  gums  assume  various 
shades  of  red ;  the  membrane  of  the  nucleus  stains 
bluish ;  resins,  pure  blue  (the  cuticle  also  colors 
blue  in  many  colleters).  Tannin  assumes  a  foxy 
color;  the  cell -wall  stains  pale  violet,  deeper  if 
it  contains  lignin,  reddish  if  it  is  more  gelatinous. 
Bast  cells  are  stained  a  deep  red ;  sieve  tubes  and 
the  soft  bast  do  not  assume  any  intense  color, 
which  is  of  advantage  in  the  study  of  the  fibro- 
vascular  bundles  of  endogens. 


METHYL   VIOLET 

Has  been  recommended  as  a   staining  agent  for 
bacteria  by  Koch,1  whose  methods  we  give. 

A  few  drops  of  a  concentrated  tincture  of  me- 
thyl violet  are  added  to  15  to  20  gm.  of  distilled 
water,  so  that  this  is  deeply  colored.  With  a 
small  pipette  a  couple  of  drops  of  this  are  placed 
upon  the  film  of  bacteria  to  be  colored,  where  the 
fluid  is  allowed  to  flow  back  and  forth  until  it  is 
thought  that  the  specimen  is  sufficiently  stained. 
After  a  little  practice  it  is  easy  to  determine  the 
proper  concentration  for  the  fluid,  and  the  length 
of  time  it  requires  to  act.  If  it  is  too  weak  the 
bacterian  film  loosens  from  the  glass,  while  if  it 

l  Koch :  Cohn's  Beitr.  z.  Biol.  der  Pfl.,  II.,  p.  406. 


COLORING   AGENTS.  6 1 

contains  too  much  of  the  coloring  matter  the  mass 
in  which  the  bacteria  lie  stains  too  deeply.  After 
the  process  of  staining  is  finished,  the  preparation 
is  rinsed  with  distilled  water,  or  with  a  ten  per 
cent,  solution  of  potassic  acetate. 

After  lying  for  half  an  hour  exposed  to  the  air, 
the  slide  is  ready  for  mounting  in  balsam.  Gly- 
cerine cannot  be  used,  as  it  removes  the  color. 
Preparations  which  are  to  be  photographed  should 
be  mounted  in  a  fifty  per  cent,  solution  of  potassic 
acetate  and  sealed  air-tight. 

The  coloring  matter  is  so  quickly  taken  up  by 
the  bacteria  that  we  have  in  it  a  useful  reagent 
for  these  organisms,  which  might  be  easily  con- 
founded with  small  oil  globules  or  other  very 
minute  rounded  bodies. 


ANILINE   BLUE. 

Wilhelm T  and  Russow 2  have  recommended  an 
aqueous  solution  of  this  dye  for  staining  the  cal- 
lous-plates of  sieve-tubes.  After  the  sections  have 
been  submitted  to  the  action  of  the  dye  they  are 
rinsed  in  water.  The  protoplasm  colors  violet- 
blue  ;  the  nuclei,  deep  indigo.  Cellulose  mem- 
branes assume  a  blue  color,  while  the  callous- 
plates  become  azure.  Preparations  mounted  in 
glycerine  change  in  a  few  days,  so  that  only  the 

1  Wilhelm :  Siebrohren,  1880,  p.  36. 

2  Russow :  Sitzber.  d.  naturf.  Ges.,  Dorpat,  1881,  p.  63. 


62  MICRO-CHEMICAL  REAGENTS. 

nuclei  and  callous-plates  remain  colored.  The 
latter  are  very  evident,  because  of  their  considera- 
ble refractive  power.  Such  preparations  remain 
unchanged  for  several  months. 

I  can  recommend  this  blue  for  giving  a  very 
intense  color  to  bacteria.  It  is  used  precisely 
like  methyl  violet.  The  preparations  should  be 
mounted  in  pure  Canada  balsam,  as  the  chloroform 
solution  removes  the  coloring  matter. 

[More  recently  methyl  blue  has  been  used  for 
detecting  the  Bacillus  of  consumption.  The  tu- 
berculous matter  is  dried  in  a  thin  film  on  a  cover- 
glass  in  the  usual  way,  and  then  floated  for  twenty- 
four  hours  on  a  fluid  composed  of  i  part  saturated 
methylene  blue  in  alcohol,  2  parts  10  per  cent, 
potash,  200  parts  distilled  water.  After  rinsing 
it  is  submitted  to  the  short  action  of  a  few  drops 
of  vesuvin.  Only  the  bacteria  retain  the  blue 
color.  —  W.  T.] 

ANILINE  BROWN 

Is  used  in  the  same  way  as  methyl  violet.1  It  is 
better  than  the  latter  for  specimens  which  are  to 
be  photographed ;  but  the  preparations  must  be 
mounted  in  glycerine,  since  the  color  is  removed 
by  potassic  acetate.  A  concentrated  solution  in 
equal  parts  of  glycerine  and  distilled  water  is  rec- 
ommended as  a  staining  fluid,  the  superfluous 
color  being  washed  away  with  glycerine. 

1  Koch:  Cohn's  Beitr.,  II.,  p.  406. 


COLORING   AGENTS.  63 


ANILINE   GREEN  (Methyl  Green) 

Has  been  recommended  by  Hanstein  for  staining 
chlorophyll  grains,  to  which  it  imparts  a  deep 
green  color,  different  from  their  natural  tint. 

Treub  J  recommends  it  for  staining  the  nucleus. 
Nuclei  which  are  not  in  process  of  division  are 
colored  pale  green.  In  those  which  are  dividing 
the  so-called  nuclear-plates  assume  an  evident 
green  color.  Strasburger2  has  used  it  for  the 
same  purpose  in  combination  with  I  per  cent, 
acetic  acid. 

[For  the  use  of  iodine-green  in  double  staining 
cell-walls  for  convenience  in  class  demonstrations, 
see  Ammonia  Carmine.] 

MAGENTA. 

[Dr.  Gibbs  recommends  magenta  and  chrysoidin 
for  staining  consumptive  sputum.  Three  fluids 
are  required:  A — Magenta  crystals,  2  gm. ;  pure 
anilin,  3  gm.;  alcohol  (s.g.  830),  20  cc.;  distilled 
water,  20  cc. ;  B  —  A  saturated  solution  of  chrys- 
oidin in  distilled  water,  to  which  a  crystal  of 
thymol  has  been  added  to  prevent  deterioration  ; 
C  —  Dilute  nitric  acid  (1:2).  The  sputum,  after 
being  thoroughly  dried  on  the  cover-glass,  is 
floated  on  A  for  15-20  minutes,  then  washed  in 

1  Treub:   Sur  des  cellules  ve*getales  &  plusietirs  noyaux. — Archives 
Neerlandaises,  1880,  T.  XV. ;  pp.  7,  17  of  the  Separate. 

2  Strasburger :  Zellbild.  und  Zdltheil.,  3d  ed.,  1880. 


64  MICRO-CHEMICAL  REAGENTS. 

C  till  the  color  disappears,  rinsed  in  pure  water, 
which  restores  a  little  of  the  color,  floated  on  B 
a  few  moments,  transferred  to  absolute  alcohol, 
and  finally  dried  and  mounted  in  Canada  balsam. 
The  Bacillus  of  consumption  is  stained  by  the 
magenta,  which  does  not  color  putrefactive  bac- 
teria, and  so  differentiated  in  the  brown  sputum.1 
—  W.  T.] 


The  above  coloring  matters  are  those  which  are 
most  commonly  employed  in  micro-chemistry,  and 
.which  give  the  best  results.  There  are  some 
others  which  are  used  for  various  purposes,  but 
they  may  be  omitted  here. 

1  The  Lancet,  Aug.  5, 1882. 


APPENDIX  TO  PART  I. 


MOUNTING  MEDIA. 

Before  this  section  is  closed  the  most  important 
substances  used  in  mounting  botanical  specimens 
should  be  noticed.  In  many  cases  the  student 
must  learn  by  trial  the  best  medium  for  a  prepara- 
tion. Still  there  are  a  number  of  substances 
which  are  known  to  be  so  well  adapted  for  the 
preservation  of  very  different  objects  that  they 
should  be  tried  first. 

GLYCERINE  is  excellent  for  nearly  all  botanical 
preparations.  The  Florideae  and  Diatoms,  how- 
ever, usually  require  preservation  in  other  media ; 
the  former  since  their  cell-walls  often  swell  greatly 
in  this  fluid,  especially  if  they  have  not  been  pre- 
viously anhydrated  with  absolute  alcohol ;  the 
latter  because  their  structure  does  not  appear  in 
it  with  the  required  degree  of  distinctness.  Bac- 
teria, also,  become  so  transparent  in  glycerine, 
and  in  particular  if  they  have  not  been  stained,  as 
to  be  almost  indistinguishable. 

[FARRANT'S  SOLUTION,  which  is  employed  some- 
what in  animal  histology,  is  a  good  substitute  for 
glycerine  in  mounting  many  vegetable  tissues. 
Frey's  formula  is :  Equal  parts  of  gum  arabic, 
glycerine,  and  a  saturated  solution  of  arsenious 
acid.  If  the  slide  is  allowed  to  lie  a  day  or  two 

65 


66  APPENDIX   TO   PART  I. 

before  being  sealed,  the  gum  hardens  at  the  edge 
of  the  cover,  and  so  aids  in  fastening  it.  This 
medium  does  not  render  sections  so  transparent 
as  pure  glycerine. 

GLYCERINE  AND  ACETIC  ACID  in  equal  parts 
make  a  convenient  preservative  for  many  fungi 
and  other  preparations.  The  fluid  should  be  boiled 
and  filtered  to  remove  mold  spores  and  other 
impurities. 

Like  other  fluid  media,  this  requires  the  employ- 
ment of  cells  of  some  sort, — the  usual  ring  of 
asphalt  or  the  wax  cell,  if  firmly  fixed,  answering 
very  well.  In  covering  such  a  cell  it  is  best  to 
lower  the  cover  gradually  from  one  side,  so  that 
any  superfluous  fluid  which  is  forced  out  shall 
penetrate  between  the  cell  and  cover-glass  only 
at  one  side,  where  it  must  be  carefully  wiped  off 
with  blotting-paper  before  the  cell  is  sealed. 

A  neat  and  useful  cell  may  be  made  by  placing 
three  small  balls  of  white  wax  on  the  slide,  at 
equal  distances  apart,  just  within  the  line  where 
the  edge  of  the  cover  is  to  come,  and  flattening 
them  to  the  requisite  thickness  by  pressing  the 
slide  against  any  flat  body  covered  by  a  piece  of 
muslin  or  paper.  A  drop  of  the  mounting  fluid 
is  put  in  the  centre,  and  the  object  arranged  in  it 
as  it  is  to  remain,  after  which  the  cover-glass  is 
slightly  warmed,  placed  in  position,  and  gently 
pressed  against  the  wax  supports  by  the  handle  of  a 
dissecting  needle  or  other  convenient  object.  Any 


MOUNTING  MEDIA.  6/ 

fluid  which  has  escaped  beyond  its  edge  is  neatly 
removed  with  bits  of  bibulous  paper,  and  a  mod- 
erately-heavy ring  of  benzole-balsam  is  painted 
round  the  cover.  When  this  is  dry  a  coating  of 
asphalt  may  be  added  to  toughen  the  cell  if  de- 
sired. With  a  little  practice  one  learns  to  esti- 
mate the  quantity  of  fluid  needed  in  a  given  case, 
so  that  very  little  is  forced  out,  and,  the  slide  being 
perfectly  dry  and  clean,  except  at  the  one  point 
of  escape,  the  cell  which  is  built  up  adheres 
well  — W.  T.] 

GLYCERINE  JELLY.  —  Nordstedt's r  directions  for 
the  preparation  of  this  substance  (for  mounting 
algae)  are  as  follows :  One  part  of  pure  gelatine  is 
dissolved  in  three  parts  of  hot  distilled  water  and 
four  parts  of  glycerine.  To  prevent  moulding,  a 
small  piece  of  camphor  or  a  drop  of  carbolic  acid 
is  added.  The  mass  hardens  on  cooling,  and  must 
be  slightly  warmed  when  needed  for  use. 

Kaiser2  gives  the  following  recipe  :  One  part  by 
weight  of  the  best  French  gelatine  [Cox's  gelatine 
is  equally  good]  is  soaked  in  six  parts  of  distilled 
water  for  two  hours,  seven  parts  of  chemically 
pure  glycerine  are  added,  and  I  gm.  of  carbolic 
acid  is  added  to  100  gms.  of  the  mixture.  The 
whole  is  then  heated  ten  to  fifteen  minutes,  mean- 


1  Om  anvandandet  af  gelatinglycerin  vid  undersbkning  og  preparering 
af  Desmidieer.  —  Botaniska  Notiser,  1876,  No.  2. 

2  Botanisches   Centralblatt,    1880,  No.  i,  p.  25. —  Cf.  also   Glycerin- 
Gelatine  for  Mounting. — Journ.of  Royal  Micr.  Soc.,  1880,  Vol.  III.,  p.  502. 


68  APPENDIX   TO    PART   I. 

time  being  constantly  stirred,  until  the  fluid  has 
become  clear,  after  which  it  is  filtered  through 
glass  wool. 

This  glycerine,  jelly,  which,  in  a  thin  layer,  is 
completely  transparent  and  as  clear  as  water,  is 
useful  for  preparations  that  are  to  lie  immovably 
under  the  -cover-glass,  but  which  are  too  small  to 
be  held  by  the  pressure  of  the  latter.  Pollen 
grains,  starch,  yeast-cells,  spores,  and  especially 
unicellular  algae,  e.  g.  Desmidiacae,  should  be 
mounted  in  this  medium.  If  it  is  desired  to  pre- 
serve the  structure  of  the  protoplasm  and  the 
arrangement  of  the  chlorophyll  bodies  as  far  as 
possible,  the  plants  must  be  previously  hardened 
by  immersion  in  an  aqueous  solution  of  perosmic 
acid  (i  :  800)  or  in  absolute  alcohol.  After  being 
hardened  the  preparations  are  placed  in  dilute 
glycerine  before  being  mounted  in  glycerine  jelly. 
When  this  is  cold  the  cover-glass  is  sealed. 

[Taking  advantage  of  the  insolubility  of  gela- 
tine which  has  been  acted  upon  by  potassium 
bichromate  and  exposed  to  the  light,  Dr.  Goodale 
has  recommended  lightly  painting  the  edge  of  the 
cover-glass  with  a  solution  of  this  salt  in  place  of 
the  usual  ring  of  varnish.  — W.  T.] 

CHLORIDE  OF  CALCIUM  is  often  used  in  aqueous 
solution  for  many  kinds  of  preparations,  —  starch, 
however,  excepted.  One  part  of  chloride  of  cal- 
cium to  three  of  distilled  water,  with  a  trace  of 
hydrochloric  acid  (to  prevent  crystallization  under 


MOUNTING  MEDIA.  69 

the  cover-glass),  has  been  recommended.  There 
are,  however,  so  many  unpleasant  features  about 
the  use  of  this  preservative  that  I  prefer  not  to 
use  it. 

ACETATE  OF  POTASSIUM.  —  A  concentrated  aque- 
ous solution  is  used  for  preparing  algae  for  ana- 
tomical preparations  as  well  as  for  the  preservation 
of  bacteria  stained  in  methyl-violet.  Slides  should 
lie  twenty-four  hours  before  the  covers  are  sealed 
down. 

[MONOBROMO-NAPHTHALIN  is  employed  by  Holier 
in  mounting  diatoms  for  test  objects,  because  of 
its  great  refractive  power.  The  markings  of  the 
frustules  come  out  nearly  as  well  as  in  dry  mounts, 
while  the  specimens  present  a  much  better  appear- 
ance.1 

PHOSPHORUS  is  also  used  for  the  same  class  of 
objects,  giving,  it  is  said,  even  better  results. — 
W.  T.] 

CANADA  BALSAM,  which  liquefies  when  slightly 
warmed,  is  much  used  in  mounting  diatoms,  the 
fine  structure  of  their  frustules  being  brought  out 
very  clearly  by  it.  Instead  of  the  pure  balsam,  a 
concentrated  solution  in  ether  or  chloroform,  which 
is  far  cleanlier,  may  be  used. 

[For  most  purposes  benzole-balsam  is,  by  some, 
preferred  to  either  of  these  solutions.  It  is  pre- 
pared by  exposing  commercial  "balsam  of  firs"  to 
gentle  heat  in  a  shallow  dish,  loosely  covered  to 

l  Cf.  Journ.  Roy.  Micr.  Soc.,  1880,  III.,  p.  1043. 


7O  APPENDIX   TO   PART   I. 

exclude  dust,  until  all  water  is  evaporated,  and  the 
balsam  becomes  perfectly  hard  on  cooling.  It  is 
then  dissolved  in  enough  pure  benzole  to  make  a 
thin  solution,  and  filtered.  The  usual  directions 
advise  subsequent  evaporation  to  the  consistency 
of  cream,  but  it  may  be  satisfactorily  employed  for 
most  purposes  in  a  much  more  fluid  form.  Glass 
stoppers,  which  are  always  advisable  for  reagent 
bottles,  are  indispensable  for  bottles  intended  to 
hold  this  or  Dammar.  Sections  may  be  mounted 
in  this  solution  directly  from  benzole,  or  from  either 
of  the  commonly  used  clearing  fluids  —  clove  oil  or 
turpentine. — W.  T.] 

Only  such  things  as  contain  little  water  should 
be  mounted  in  balsam.  If  the  objects  are  delicate 
and  contain  much  water,  they  must  be  anhydrated 
in  absolute  alcohol,  or  dried  in  the  air,  and  when 
it  is  necessary  they  should  be  cleared  in  clove  oil 
before  mounting.  Although  balsam  preparations 
cannot  dry  up,  or  move  about  after  the  medium 
has  once  hardened,  they  need  to  be  sealed,  as 
a  jar  may  at  any  time  loosen  the  cover.  Such  a 
partial  or  complete  loosening  is  easily  detected  by 
the  appearance  of  Newton's  rings.1 

1  Literature  of  Mounting  Media.  —  Dippel .  Das  Mikroskop,  I.,  p.  470, 
et  seq.  Schacht:  Mikroskop,  1855,  p.  28.  Sanio :  Bot.  Zeitung,  1863,  p. 
359.  Koch:  Conn's  Beitr.  z.  Biol.  d.  Pfl.,  Bd.  II.,  p.  407.  Bachmann: 
Dauerpraparate,  1879.  Frey:  Mikroskop,  pp.  122,  125.  Harting:  Mikro- 
skop, III.,  p.  409.  Pelletan :  Le  Microscope,  1876,  p.  178,  et  seq. 


CEMENTS. 


CEMENTS. 

A  large  number  of  compositions  have  been  pro- 
posed for  use  as  cements  for  mounting  prepara- 
tions. As  only  a  few  of  these  appear  to  me 
deserving  of  attention,  I  do  not  hesitate  to  add  a 
few  words  on  this  subject  to  the  digression  already 
made  in  behalf  of  mounting  media. 

Cements  should  adhere  to  glass  strongly,  so  as 
to  be  both  air  and  water-tight.  They  should  be 
unaffected  by  the  mounting  fluid,  and  should  not 
crack  with  age. 

Those  which  I  would  recommend,  from  my  own 
experience,  are  asphalt  varnish  (Brunswick  black), 
which  can  be  bought  ready  for  use,  and  is  made  by 
dissolving  asphalt  in  linseed  oil  or  turpentine ;  pre- 
pared gold-size  (a  sort  of  Copal  varnish)  ;  and  an 
alcoholic  solution  of  Holmblad's  best  sealing  wax. 
All  these  substances  should  have  a  rather  ropy 
consistency,  and  must  not  harden  too  quickly. 
They  are  applied  with  a  small  camel's-hair  pencil. 
The  safest  sealing  is  effected  by  applying  a  layer 
of  asphalt  first,  and  covering  this  with  the  sealing- 
wax  solution  when  it  is  half-hard. 

In  my  opinion  the  following  new  composition  is 
to  be  recommended.  We  have  tried  it  in  the 
Copenhagen  laboratory,  and  so  far  are  pleased  with 
it.  50  gm.  Canada  balsam ;  50  gm.  shellac ;  50 
gm.  absolute  alcohol,  and  100  gm.  ether  are  mixed, 


72  APPENDIX   TO   PART   I. 

filtered,  and  evaporated  over  the  water-bath  to  the 
consistency  of  thick  syrup.  I  call  this  the  "  Gram- 
Riitzou  "  varnish,  after  its  discoverers. 

I  have  not  personally  tested  the  mastic  cement 
(Maskenlack,  No.  3),  recommended  by  German 
investigators,  nor  Ziegler's  and  other  foreign  com- 
positions. 1  can,  however,  recommend  the  so- 
called  Japan  varnish,  which  seems  to  me  very  use- 
ful for  the  first  coat,  when  applied  similarly  to 
asphalt. 

In  the  laboratory  at  Rome  a  cement  is  used, 
prepared  by  dissolving  with  the  aid  of  heat  100 
parts  gum  dammar  in  100  parts  benzole,  and  add- 
ing 60  parts  ivory  black  ground  with  oil. 

An  alcoholic  solution  of  shellac  can  be  used  for 
fastening  the  label  to  the  slide,  as  gums  and  other 
adhesive  substances  do  not  adhere  well  to  the 
glass.  [Fish  glue  and  the  liquid  glue  made  with 
acetic  or  nitric  acid  are  useful  for  this  purpose, 
though  rather  disagreeable  to  use. — W.  T.] 


PART  II. 
VEGETABLE   SUBSTANCES 

AND  THE 

METHODS   OF   RECOGNIZING  THEM. 


PART  II. 


VEGETABLE    SUBSTANCES 


METHODS   OF   RECOGNIZING  THEM. 


[The  reader  is  requested  to  consult  the  principal  literature  in  the  first 
section,  under  the  different  reagents.] 

CELLULOSE. 

Pure  cellulose  is  colored  violet  by  chlor-iodide  of 
zinc,  blue  by  iodine  and  sulphuric  acid,  and  brown, 
or  yellowish  brown,  or  even  yellow,  by  an  aqueous 
or  alcoholic  solution  of  iodine.  On  the  addition 
of  water  to  membranes  which  have  been  dried 
after  treatment  with  iodine,  they  often  become 
clear  blue.  Young  cellulose  frequently  colors  blue 
with  iodine  and  sulphuric  acid,  only  after  treatment 
with  hydrochloric  acid,  or  after  being  compressed 
strongly  under  the  cover-glass.1 

The  cellulose  of  the  seeds  of  Paeonia  colors 
blue  in  iodine  dissolved  in  iodide  of  potassium. 

1  Richter:  Sitzb.  wien.  Akad.,  1881,  Bd.  LXXXIII. 

75 


76  VEGETABLE   SUBSTANCES. 

Cellulose  swells  in  an  aqueous  solution  of  po- 
tassic  hydrate,  as  well  as  in  the  so-called  min- 
eral acids,  and  the  stratification  of  the  cell-wall  is 
frequently  rendered  much  sharper  and  more  dis- 
tinct as  a  result.  It  is  dissolved  by  concentrated 
sulphuric  acid  with  the  formation  of  amyloid,  and 
in  cuprammonia  without  change.  From  this  solu- 
tion it  can  be  precipitated  by  the  use  of  absolute 
alcohol. 

Cellulose  is  colored  with  unequal  intensity  by 
the  various  aniline  colors.  Alcannin  and  car- 
mine, on  the  other  hand,  are  inactive,  or  nearly  so. 
After  heating  with  concentrated  potassic  hydrate, 
young  cellulose  is  not  colored  by  cupric  sulphate  ; 
but  when  somewhat  older  it  is  colored  pale  blue  by 
this  reagent.  Grenadier's  alum  carmine  colors  it 


LIGNIN. 

This  substance  probably  consists  of,  i,  vanillin  ; 
2,  coniferin(?)  ;  3,  some  gum  ;  4,  a  substance  which 
is  colored  yellow  by  hydrochloric  acid;  and  5,  the 
wood-gum  of  Thomsen.  It  is  thus  seen  to  be  a 
very  complex  substance.1 

Lignified  cell-walls  are  colored  yellow  by  chlor- 
iodide  of  zinc.  Concentrated  sulphuric  and  chro- 
mic acids  dissolve  them,  but  they  are  insoluble  in 
cuprammonia  and  the  Schultze  maceration  fluid. 
After  treatment  with  an  aqueous  solution  of  cupric 

l  Max  Singer:  Anzeiger  des  kais.  Akad.,  Wien,  1882,  No.  u. 


INTERCELLULAR  SUBSTANCE.         77 

sulphate,  the  addition  of  warm  concentrated  potas- 
sic  hydrate  often  colors  them  brown.  An  aqueous 
solution  of  sulphate  of  aniline  (naphthalidin  or 
toluidin),  followed  by  saturation  with  dilute  sul- 
phuric acid,  colors  them  a  beautiful  yellow  (Wies- 
ner's  reaction).  Frequently  this  reaction  is  ob- 
tained without  the  use  of  any  acid.  All  membranes 
which  contain  lignin  assumes  an  intense  and  very 
beautiful  rose  color,  due  to  the  presence  of  vanillin, 
when  treated  with  hydrochloric  acid  and  phloro- 
glucin.  The  same  reaction  is  given  by  this  acid  in 
combination  with  an  extract  of  cherrywood  (Xylo- 
filin),  but  the  color  is  more  violet.  On  the  other 
hand,  a  bluish-green  color  appears  after  the  succes- 
sive action  of  hydrochloric  acid  and  phenol. 

All  aniline  colors  are  taken  up  with  avidity  by 
lignified  membranes.  Indol  gives  them  an  in- 
tense red  color.  Grenadier's  alum  carmine  does 
not  color  them.  A  weak  aqueous  solution  of  eosin 
produces  no  color  after  short  action,  though  the 
same  is  true  of  pure  cellulose.  Lignified  mem- 
branes lose  the  so-called  incrusting  substances  by 
being  heated  with  alkalies,  concentrated  nitric  acid, 
or  the  Schultze  maceration  fluid,  after  which  the 
cellulose  reaction  can  be  produced  by  the  proper 
reagents. 

INTERCELLULAR    SUBSTANCE   (''Middle   Lamella"). 

Insoluble  in  concentrated  sulphuric  acid,  cu- 
prammonia,  and  dilute  chromic  acid  ;  soluble  with 


78  VEGETABLE   SUBSTANCES. 

difficulty  in  concentrated  chromic  acid ;  soluble  in 
the  Schultze  maceration  fluid  (which  is  specially 
important  in  the  investigation  of  wood),  and  occa- 
sionally in  hot  potash,  sometimes  even  in  boiling 
water. 

The  intercellular  substance  is  easily  and  strongly 
colored  by-  the  aniline  dyes.  With  chlor-iodide  of 
zinc  it  assumes  a  yellow  color.  Hot  nitric  acid 
and  ammonia  give  it  a  beautiful  yellow  color.1 


SUBERIN, 

The  constituent  of  cork,  is  insoluble  in  concentrated 
sulphuric  acid  and  cuprammonia,  and  very  resistant 
to  chromic  acid.  Treated  with  boiling  potash, 
corky  membranes  secrete  peculiar  ochre-yellow 
granular  masses,  and  when  heated  with  nitre  acid 
and  chlorate  of  potassium  they  form  masses  of 
eerie  acid,  which  are  soluble  in  alcohol,  ether, 
benzole  and  chloroform. 

The  walls  of  cork  cells  are  colored  yellow  by 
chlor-iodide  of  zinc.  Grenacher's  alum  carmine 
does  not  stain  them. 

Olivier  indicates  the  following  method  of  color- 
ing corky  membranes.  Sections  of  the  tissue  to 
be  studied  are  laid  in  a  solution  of  fuchsin  in 
equal  parts  of  alcohol  and  water,  which  is  taken 

1  Solla:  Beitr.  zur  Kenntniss  d.  chem.  und  phys.  Beschaffenheit  der 
Intercellularsubstanz.  —  Oesterr.  botan.  Zeitschr.,  Nov.  1879.  Hohnel :  Mit- 
tellamellc  der  Holzelemente  u.  d.  Hoftiipfelschliessmembran.  —  Bot.  Zeitung, 
1880,  No.  26. 


FUNGUS-CELLULOSE.  79 

up  by  the  entire  preparation.  By  after-treatment 
with  absolute  alcohol,  the  coloring  matter  is  re- 
moved from  the  cellulose  portions,  remaining  only 
in  those  which  are  corky.1 


FUNGUS-CELLULOSE. 

In  1866  De  Bary  gave  this  name  to  the  sub- 
stance composing  the  cell-walls  of  fungi.2  Pre- 
viously (1852)  Schacht  had  shown3  that  these 
walls  were  very  resistant  to  micro-chemical  re- 
agents, and  especially  that  the  reaction  character- 
istic of  pure  cellulose  seldom  succeeds.  Since 
then,  the  names  fungine  and  metacellulose  have 
been  given  to  this  doubtful  substance.  Recently 
Richter  has  succeeded  in  showing  that  the  walls 
of  hyphae  are,  in  reality,  formed  of  cellulose  as  a 
fundamental  substance,4  its  detection  being  ren- 
dered difficult  by  the  presence  of  infiltrated  mat- 
ters, —  possibly  of  a  protein  nature. 

After  fungus  tissues  have  been  treated  with  con- 
centrated potash,  frequently  renewed,  for  several 
weeks,  and  especially  after  they  have  been  finally 
boiled  in  this  fluid,  they  assume  a  blue  color  with 

1  Olivier :  Note  sur  le  systeme  te"gumentaire  des  racines  chez  les  Pha- 
ne*rogames. —  Bull.  Soc.  hot.  de  France,  1880,  T.  XXVII.,  pp.  234-235. 

2  De  Bary :  Morphol.  u.  Physiol.  d.  Pilzen,  Flechten  u.  Myxomyceten, 
p.  7,  et  seq. 

3  Schacht :  Pflanzenzelle,  p.  9. 

4  Richter :  Beitr.  z.  genaueren  Kenntniss  d.  chem.  Beschaffenheit.  d. 
Zellmemb.  bei  den  Pilzen.  —  Sitzber.  wiener  Akad.,  1881,  Bd.  LXXXIL, 
Abth.  i. 


8O  VEGETABLE   SUBSTANCES. 

the  reagents  used  for  detecting  pure  cellulose.1 
There  is,  therefore,  no  reason  for  considering 
"metacellulose  "  as  a  distinct  substance.  It  should 
be  looked  upon  simply  as  a  modification  similar  to 
that  of  wood  and  bark. 

The  paraphyses  and  asci  of  lichens  are,  as  a 
rule,  colored  blue  by  iodine,  as  are  the  hyphae  of 
the  medullary  layer  in  some  cases.  This  is  to  be 
attributed  to  the  presence  of  lichenin,  —  a  sub- 
stance characteristic  of  these  plants. 


PROTEIN  SUBSTANCES 

Are  easily  characterized  by  the  brown  color  which 
is  imparted  to  them  by  iodine,  the  rosy  red  which 
they  assume  when  acted  upon  by  Millon's  reagent, 
especially  after  gentle  warming,  and  the  yellow 
which  is  produced  by  nitric  acid 2  either  alone  or 
in  combination  with  ammonia.  After  lying  for 
about  twelve  hours  in  corrosive  sublimate  in 
alcohol,  they  form  a  compound,  insoluble  in  water, 
which  is  especially  interesting  in  preparations  of 
aleuron  grains.  Protein  compounds  become  violet 
when  treated  with  Trommer's  reagent ;  sugar  and 
sulphuric  acid  (Raspail's  reagent,  1833)  color  them 
red.  The  imbibition  and  condensation  of  different 
coloring  matters,  e.  g.  cochineal,  carmine,  a  solu- 

1  Van  Tieghem:  Traite  de  botanique,  1882,  p.  569. 

2  Discovered  in   1686    [?]   by  Glauber   (Explicatio    miraculi   mundi). 
Mulder  has  given  the  yellow  compound  the  name  "  Xanthoproteic  acid" 


PROTOPLASM.  8 I 

tion  of  aniline  blue  in  water,  etc.,  is  characteristic 
of  protein  substances.1 

For  the  preparation  of  aleuron-grains  the  fol- 
lowing method  is  employed  in  the  Copenhagen 
laboratory :  The  sections  are  treated  for  several 
days  with  a  five  per  cent.-  alcoholic  solution  of 
corrosive  sublimate,  according  to  Pfeffer's  plan. 
They  are  then  colored  by  an  aqueous  solution  of 
eosin,  and  mounted  for  study  in  acetate  of  potas- 
sium and  water  in  equal  parts.  The  crystalloids 
are  thus  rendered  very  distinct ;  and,  if  the  eosin 
solution  has  not  been  too  concentrated,  they  com- 
monly assume  a  red  shade,  different  from  that  of 
the  ground  mass. 


PROTOPLASM. 

Protoplasm  is  the  living  portion  of  the  cell. 
The  chemical  cause  of  this  life  —  if,  indeed,  such 
chemical  cause  exist  —  has  not  yet  been  discov- 
ered ;  but  it  appears  as  if  some  steps  have  been 
taken  toward  the  solution  of  this  interesting  prob- 
lem. Quite  recently  a  reaction  for  living  proto- 
plasm has  been  discovered.  In  this  state  it  con- 
tains free  aldehyde,  which  does  not  exist  in  dead 
protoplasm.  This  substance  precipitates  metallic 
silver  from  even  an  extremely  dilute  alkaline  solu- 

1  Cf.  Vines :  Chemical  composition  of  aleuron-grains.  —  Royal  Society 
of  London,  May  13, 1880.  —  Nature,  1880,  vol.  22,  No.  552,  p.  91.  — Journ. 
of  the  Royal  Mien  Soc.,  1880,  Vol.  III.,  p.  667. 


82  VEGETABLE    SUBSTANCES. 

tion  of  the  nitrate.  The  protoplasm  is,  therefore, 
colored  a  pronounced  black.  (Cf.  p.  41.) 

Since  protoplasm  is  a  mixture  of  different 
protein  compounds,  it  gives  their  characteristic 
reaction.  Living  protoplasm  does  not,  however, 
imbibe  coloring  matters  as  dead  protoplasm  does. 
A  solution '  of  caustic  potash  or  concentrated 
ammonia  water  increases  its  transparency ;  acetic 
acid  produces  the  opposite  effect.  Absolute  alco- 
hol affects  it  very  characteristically,  rapidly  har- 
dening it.  This  is  of  especial  value  in  various  in- 
vestigations of  protoplasmic  structure,  the  changes 
which  occur  in  the  embryo  sac,  the  division  of 
the  nucleus,  etc.  An  aqueous  solution  of  peros- 
mic  acid,  even  when  very  dilute  (i  :  800),  has  a 
similar  effect. 

Aqueous  solutions  of  sugar,  table-salt,  alcohol, 
glycerine,  etc.,  by  the  abstraction  of  water,  cause 
the  protoplasm  of  cells  to  contract  and  separate 
from  the  wall.  This  frequently  happens  without 
killing  the  protoplasm,  though  this  of  course  is  not 
the  case  with  alcohol  unless  greatly  diluted  with 
water. 

The  metaplasm  of  Hanstein,1  i.  e.  that  part  of 
the  protoplasm  which  holds  the  formative  material, 
is  colored  almost  scarlet  by  Hanstein's  aniline 
violet.  De  Bary's  epiplasm,2  a  special  modifica- 

1  Organe  der  Harz-  und  Schleimabsonderung.  —  Bot.  Zeitung,   1868, 
p.  709. 

2  Morphol.  u.  Phys.  d.  Pilze  etc.  —  Hofmeister's  Handb.,  p.  103. 


PROTOPLASM.  83 

tion  of  protoplasm  in  the  asci  of  ascomycetes  [con- 
taining much  glycogen],  assumes  a  deep  reddish- 
brown  or  violet-brown  color  with  even  a  very  dilute 
solution  of  iodine. 

The  preparation  of  the  nucleus,  which  has  been 
so  zealously  studied  of  late,  has  formed  the  subject 
of  many  contributions.1  Acetic  acid,  alcohol,  and 
perosmic  acid  differentiate  it  sharply,  and  are 
therefore  used  to  render  it  evident.  Coloring 
matters  are  taken  up  and  condensed  by  the  nu- 
cleus. The  effect  of  haematoxylin,  aniline  green, 
Grenacher's  alum-carmine,  ammonia-carmine,  and 
picro-carmine,  is  to  color  the  nucleus  deeper  than 
the  rest  of  the  protoplasm  in  the  cell.  Iodine  acts 
in  exactly  the  same  manner.  Successive  treat- 
ment with  alcohol,  picro-carmine,  and  glacial  acetic 
acid  is  recommended  by  Maupas. 

Among  the  various  substances  of  the  protein 
group,  nuclein  has  been  especially  studied  by 
Zacharias 2  and  others.  To  detect  it,  immerse  the 
preparation  for  an  hour  in  a  mixture  of  one  volume 
of  a  ten  per  cent,  solution  of  the  yellow  ferro- 
cyanide  of  potassium  in  distilled  water,  and  two 
volumes  of  acetic  acid,  prepared  by  diluting  the 
glacial  acid  with  an  equal  bulk  of  water.  Wash 
the  sections  in  sixty  per  cent,  alcohol.  The 

1  Strasburger,  Hanstein,  Ranvier,  Treub,  Schmitz,  and  others  have 
done  much  in  this  direction.     Cf.  further.  —  Treub :  Actes  du  Congres  In- 
ternational des  Botanistes,  etc.,  a  Amsterdam  en  1877,  Leide,  1879,  P-  14^- 
—  Maupas:  Comptes  rendus,  July  1879,  Vol.  LXXXVIIL,  p.  250. 

2  Zacharias:  Bot.  Zeitung,  1881,  p.  169;  1883,  p.  209. 


84  VEGETABLE   SUBSTANCES. 

masses  of  nuclein  are  characterized  by  the  blue 
color  they  assume,  which  was  discovered  by 
Hartig.1 

STARCH  2 

Is  colored  blue  by  the  tincture  of  iodine,  iodide  of 
potassium,  and  other  substances  which  contain 
free  iodine,  since,  although  the  so-called  starch 
cellulose  cannot  take  up  this  element,  granulose 
does  so.  The  blue  color  which  results  is  not  due 
to  a  chemical  compound,  but  is  rather  to  be  con- 
sidered as  the  result  of  a  (molecular  ?)  solution  of 
the  iodine  in  granulose.3  In  this  connection  we 
must  remind  the  reader  that  the  presence  of  water 
is  a  conditio  sine  qua  non  for  the  reaction.  Dry 
starch  treated  with  the  vapor  of  iodine,  or  with 
iodine  in  anhydrous  alcohol  or  chloroform,  is  only 
colored  brown  like  pure  starch  cellulose. 

All  substances  which  combine  directly  with 
iodine  destroy  the  blue  color.  Gentle  warming  in 
water  has  the  same  effect ;  but  in  cooling,  the 
starch  resumes  its  color. 

When  slowly  heated  in  water  starch  grains  swell 
considerably,  usually  after  the  temperature  has 
reached  50°  C.  In  this  way  starch  paste  is  formed, 

1  Hartig:  Bot.  Zeitung,  1854. 

2  Nageli :  Beitrage  zur  naheren  Kenntniss  der  Stark egruppe,  1874. 

3  The  compound  resulting  from  the  long  treatment  of  starch  with  salt- 
solutions  containing  an  excess  of  free  iodine  is  somewhat  different.     After 
being  raised  to  a  red  heat,  this  still  contains  about  three  per  cent,  of  iodine. 
—  Cf.  E.  Sonstadt :  Note  on  the  compound  of  starch  with  iodine.  —  Chemi- 
cal News,  1873,  Vol.  XXVIII.,  p.  248. 


STARCH.  85 

which  may  be  diffused  in  the  water,  but  does  not 
form  a  true  solution.  This  paste  gives  the  same 
reaction  as  unaltered  starch.  Solutions  of  chlo- 
ride of  calcium,  chloride  of  zinc,  potassic  hydrate, 
and  concentrated  iodide  of  potassium,  as  well  as 
the  mineral  acids,  carbolic  acid,  acetic  acid,  and 
trichloracetic  acid  cause  this  conversion  of  starch 
into  paste  in  varying  degrees,  depending  upon  their 
concentration.  The  beginning  of  the  change  may 
always  be  recognized  by  the  more  evident  stratifi- 
cation of  the  grains.  Dilute  chromic  acid  produces 
this  effect  in  a  marked  degree,  and  has  been  used 
to  demonstrate  stratification  in  the  peculiar  starch 
bodies  which  are  found  in  the  latex  of  Euphor- 
biaceae. 

Alcohol  has  an  opposite  effect,  the  stratification 
often  completely  disappearing  under  its  action. 
Cuprammonia  causes  the  grains  to  swell,  and  colors 
them  pale  blue,  but  it  does  not  change  them  into 
paste. 

For  directions  for  demonstrating  the  starch 
in  chlorophyll  bodies  the  reader  is  referred  to 
page  7. ' 

1  Crie  announces  a  new  substance  found  by  him  in  the  asci  of  Sphaeria 
Desmazierei,  Berk.,  which  he  calls  amylomycin,  and  which  is  said  to  have 
the  same  reaction  as  starch.  (Comptes  rendus,  T.  LXXXVIII.,  pp.  759, 
985.)  We  mention  this  here  merely  to  make  our  account  complete.  The 
substance  is  so  insufficiently  characterized  by  its  discoverer  that  one  may 
be  pardoned  some  doubts  as  to  its  independent  nature. 


86  VEGETABLE   SUBSTANCES. 

[GLYCOGEN. 

This  substance,  which  occurs  in  cells  of  fungi 
in  a  semi-fluid,  amorphous  form,  and  more  or  less 
intimately  united  with  albuminoid  structures,  is 
soluble  in  water  as  well  as  in  acids  and  alkalies,  but 
insoluble  in  alcohol  and  ether.  It  does  not  reduce 
Trommer's  reagent,  and  gives  no  reaction  with 
osmic  acid,  Millon's  reagent,  or  the  salts  of  iron. 
It  may  usually  be  distinguished  from  gums  and 
mucilage  by  not  forming  gelatinous  masses  in 
water. 

The  characteristic  test  for  vegetable  glycogen 
is  the  red-brown  or  mahogany  color  it  assumes 
when  treated  with  iodine  in  the  presence  of 
water,  the  latter  being  essential,  as  in  the  cor- 
responding reaction  for  starch.  On  heating  the 
preparation  the  color  fades,  reappearing  when  it 
is  cooled. 

Errera1  recommends  crushing  the  cells  to  be 
tested  in  a  drop  of  water  under  the  cover-glass, 
and  immediately  adding  the  iodine  dissolved  in 
water  containing  a  little  iodide  of  potassium. 

Vigorous  young  plants  of  Phycomyces  nitens 
and  Coprinus  evanidus  are  recommended  for  this 
test,  though  the  reaction  may  be  obtained  with 

1  Errera  :  L'Epiplasme  des  Ascomycetes  et  le  glycogene  des  V6getaux. 
—  These  presentee  pour  1'obtention  du  grade  de  Docteur  Agrege  pres  la 
Faculte  des  Sciences  de  PUniversite  de  Bruxelles,  1882  ;  Sur  le  glycogene 
chez  les  Mucorinees. —  Bull.  Ac.  roy.  de  Belgique,  Nov.  1882,  3  S6r.,  IV., 
No.  ii. 


SUGARS.  S/ 

many  fungi,  and  is  shown  especially  well  in  asci  of 
Peziza  vesiculosa,  and  various  other  ascomycetes. 
—  W.  T.] 

DEXTRIN. 

This  transformation-product  of  starch  may  be 
recognized  in  the  vegetable  cell  by  Trommer's  test 
(p.  36).  The  vermilion  precipitate  is  finely  gran- 
ular, and  shows  the  Brownian  movement  very 
plainly  (Cf.  grape  sugar). 


GRAPE  SUGAR  (Dextrose,  Glucose) 

May  be  recognized  by  either  Trommer's  or  Feh- 
ling's  test  (p.  36).  The  reaction,  which  in  general 
is  not  very  sure,  is  manifested  by  a  reddish-yellow 
precipitate  of  cuprous  oxide.  Barfoed's  test,1  i.  e. 
heating  with  an  aqueous  solution  of  neutral  acetate 
of  copper,  gives  after  long  standing  a  red  precipi- 
tate. Such  a  precipitate  is  not  formed  with  dex- 
trine. At  ordinary  temperatures  glucose  gives  a 
precipitate  with  neutral  acetate  of  copper,  while 
dextrine  remains  clear  for  a  long  time.  With  very 
dilute  alkaline  nitrate  of  silver  (i :  100,000)  glucose 
gives  a  brown  color  (p.  41). 

i  Zeitschr.  f.  anal.  Chemie,  Bd.  XII.,  p.  27.  Sachsse :  Farbst,  Kohlen- 
hydrate  u.  Proteinsubst,  1877,  p.  192.  —  I  have  not  myself  tested  this 
reaction,  but  it  is  deserving  of  mention  here,  since  macro-chemical  reactions 
are  often  useful  in  micro-chemistry. 


88  VEGETABLE   SUBSTANCES. 

CANE  SUGAR  (Saccharose). 

Cells  which  contain  this  substance  do  not  give  a 
precipitate  with  the  Trommer  reagent,  but  assume 
a  pure  deep  violet  color.  If  much  saccharose  is 
present  in  the  tissue  it  can  be  caused  to  crystallize 
out  by  the  use  of  absolute  alcohol  (p.  26). x 

INULIN  (Sinistrin,2  Syrian  therm) 

Occurs  dissolved  in  the  cell  sap,  like  the  sugars 
which  have  just  been  mentioned.  If  a  tissue  con- 
taining it  is  treated  with  alcohol  or  glycerine  the 
inulin  separates  as  sphaero-crystals,  which  are  in- 
soluble in  cold  water,  but  easily  soluble  in  water 
heated  to  5O°-55°  C,  in  dilute  acids,  and  in  cupram- 
monia.  A  tincture  of  iodine  colors  the  sphaero- 
crystals  brown  by  penetrating  into  the  fine  clefts 
and  fissures  which  they  contain  ;  when  boiled  with 
dilute  acids  or  under  pressure  inulin  is  changed 
into  levulose. 

HESPERIDIN. 

The  sphaero-crystals  of  this  substance  are  in- 
soluble in  most  acids,  glycerine,  and  absolute 

1  See  also  Kraus'  glycerine  test  (p.  28). 

2  Not  to  be  confounded  with  the  carb-hydrate  of  the  same  formula 
which  Prof.  Schmiedeberg  has  lately  described  under  this  name,  and  which 
occurs  in  the  bulb-scales  of  Urginea  Scilla.  —  Zeitschrift  f.  physiol.  Chemie, 
1879,  p.  112.  —  Bot.  Zeitung,  1879,  p.  513. — Journal  of  the  Royal  Micr. 
Society,  1879,  Vol.  II.,  p.  916. 


GUMS,    ETC.  89 

alcohol,  as  well  as  in  cold  and  boiling  water.  They 
are  easily  soluble  in  an  aqueous  or  alcoholic  solu- 
tion of  potassic  hydrate,  assuming  a  yellow  or  red- 
dish color.  They  are  also  dissolved,  but  with  more 
difficulty,  in  hot  concentrated  acetic  acid,  ammonia, 
and  the  alkaline  carbonates. 

Unripe  oranges  may  be  used  in  testing  for  this 
substance.1 

GUMS. 

At  present  we  have  no  certain  micro-chemical 
reaction  for  gums.  The  different  sorts  are  in- 
soluble in  alcohol.  They  swell  strongly  in  water 
and  are  not  colored  blue  by  iodine,  either  alone  or 
followed  by  sulphuric  acid.  Cell-walls  which  con- 
tain gum  assume  a  red  color  when  treated  with 
Hanstein's  aniline  violet. 


VEGETABLE  MUCUS2 

Is  a  comprehensive  name  used  to  designate  a  num- 
ber of  different  substances  which  are  closely 
related  to  the  gums,  but  are  still  imperfectly 
known  in  many  respects.  They  are  distinguished 
from  gums  by  the  yellow  or  blue  color  which  they 

.1  Pfeffer:  Hesperidin.  —  Bot.  Zeitung,  1874,  P-  481.  Mika:  Beitrage 
zur  Morphol.  u.  mikroskop.  Nachweisung  des  Hesperidins.  —  Magyar 
novenytany  lapok,  L,  p.  93.  Known  to  me  only  through  Just's  Jahres- 
bericht. 

2  Kirchner  and  Tollens :  Untersuchungen  iiber  den  Pflanzenschleim.  — 
Ann.  d.  Chem.  and  Pharm.,  Bd.  CLXXV.,  p.  205. 


QO  VEGETABLE    SUBSTANCES. 

assume  with  iodine,  and  the  blue  or  violet  brown 
imparted  by  iodine  and  sulphuric  acid.  Many 
of  them  swell  considerably  in  water.  Barcianu1 
gives  the  red  color  induced  in  tissues  which 
contain  mucus,  by  successive  treatment  with 
creosote,  chloride  of  tin  and  aniline  (?),  as  a  re- 
action. 

The  so-called  amyloid  (Schleiden,  1844)  must 
belong  here.2  Leguminous  amyloid  is  colored  blue 
by  iodine  in  alcohol,  and  yellow  by  iodine  in  water. 
It  is  soluble  in  dilute  alkalies  and  in  boiling  water. 

Hanstein's  aniline  violet  colors  amyloid  sub- 
stances red,  but  of  a  shade  different  from  that 
obtained  in  the  gum  and  tannin  reactions.3 


TANNIN  (Tannic  Acid). 

Cells  containing  this  substance  are  colored  deep 
blue  or  green  by  treatment  with  ferric  acetate  (p. 
42)  or  chloride  (p.  33).  They  are  colored  reddish 
brown  by  bichromate  of  potassium  (p.  40),  fulvous 
by  Hanstein's  aniline  violet  (p.  60),  and  red  or 
violet  by  dilute  chlor-iodide  of  zinc  (p.  9).  All  of 
these  reactions  demand  a  prolonged  stay  in  the 

1  BlUthenentwickelung    der     Onagraceen.  —  Schenk     and    Luerssen's 
Mittheil.  aus  der  Bot.,  II.,  Heft  i,  p.  85.     I  have  not  made  myself  familiar 
with  this  reaction. 

2  Vogel  and  Schleiden:  Amyloid.  —  Schleiden's  Beitr.  z.  Bot.,   1844, 
Bd.  I.,  VIII. 

3  Cf.,  further,  Leon-Marchand :  G61atine  produite  par  les  Algues.  — 
Bulletin  de  la  Soc.  bot.  de  France,  1879,  P-  3$7- 


PECTIN,   ETC.  91 

fluid.1     An  alkaline  solution  of   nitrate  of   silver 
(i :  10,000)  gives  a  black  color  with  tannin  (p.  41). 


PECTIN 

Often  replaces  the  intercellular  substance  or  cer- 
tain layers  of  the  cell-wall.  It  is  recognized  by  the 
swelling  of  these  layers  in  hot  water  and  alkalies, 
and  their  solubility  in  the  latter  as  well  as  in  con- 
centrated oxalic  acid  (p.  24).  With  cuprammonia 
(p.  15),  pectate  of  copper  is  formed,  and  in  thin  sec- 
tions this  remains  after  the  complete  solution  of 
the  other  parts  of  the  wall. 

ASPARAGIN 

Is  insoluble  in  absolute  alcohol  or  a  concentrated 
solution  of  itself,  but  soluble  in  water.  On  drying 
sections  which  contain  it  or  treating  them  with 
absolute  alcohol,  acicular  crystals  of  asparagin 
form  in  the  cells  or  the  fluid  about  them  (p.  45). 


CRYSTALLOIDS2 

Is  a  name  used  to  designate  protein  bodies  hav- 
ing a  crystalline  form.      They  are   characterized 

1  Hb'hiiel  (Die  Gerberrinden,  Berlin,  1880)  gives  more  particular 
information  as  to  tannin  in  its  technical  bearings.  The  very  characteristic 
reaction  of  a  decoction  of  galls  with  iron  was  known  to  Pliny,  and  was  used 
by  the  ancients  to  detect  the  adulteration  of  verdigris  with  sulphate  of  iron 
—  the  oldest  chemical  reaction. 

8  A.    Schimper:    Untersuchungen    iiber    die    Proteinkrystalloi'de    d 


UNIVERSITY    J 


Q2  VEGETABLE   SUBSTANCES. 

by  giving  the  protein  reaction,  by  their  solubili- 
ty in  ammonia,  dilute  potash,  or  acetic  acid, 
and  by  swelling  in  water.  They  are  often  insol- 
uble in  a  potash  solution  of  above  ten  per  cent. 
There  are  also  some  which  are  soluble  in  a  solu- 
tion of  table-salt  (p.  32).  As  a  characteristic 
which  is  always  useful,  though  not  of  a  chemical 
nature,  we  mention  further  the  inconstancy  of 
their  angles. 

The  red,  somewhat  doubly  refractive,  tabular 
crystalloids  of  certain  Floridese  that  have  been 
kept  in  the  herbarium  or  mounted  in  glycerine 
are  especially  noteworthy.  They  are  the  so-called 
rhodospermin  crystals  of  Cramer,  and  are  in- 
soluble in  sodic-chloride,  in  which  they  lose  their 
color. 

The  crystalloids  contained  in  protein  grains  be- 
come visible  only  after  treatment  with  warm 
glycerine. 

CAOUTCHOUC l 

Occurs  in  the  latex  of  different  plants  in  the  form 
of  small  homogeneous  globules,  which  swell  in 
ethereal  oils,  and  are  soluble  in  carbon  bisulphide, 
chloroform  and  benzole,  but  are  not  attacked  by 
dilute  acids  or  alkalies. 

Pflanzen,  Strassburg,  1879.  J.  Klein:  Krystalloide  der  Meeresalgen. — 
Flora,  1880,  p.  65.  Cohn's  Beitrage  zur  Biol.  d.  Pfl.,  1880,  III.,  p.  163 
(Crystalloids  in  the  nuclei  of  Pinguicula  alpina). 

1  Weiss:  Allg.  Bot.,  L,  p.  191.  Duchartre:  Elements  de  Bot.,  1877, 
p.  74.  . 


CHRYSOPHANIC   ACID,    ETC.  93 

CHRYSOPHANIC  ACID. 

Cells  which  contain  this  acid  are  colored  deep 
red  by  potassic  hydrate.  The  hyphae  of  lichens 
are  also  colored  red  by  the  reagent,  and  are  dis- 
solved by  it.  On  the  other  hand,  calcic  hydrate 
or  barium  water  colors  them  without  dissolving 
them.  Ammonium  carbonate  gives  no  color  with 
this  substance,  and  hydrochloric  acid  does  not 
affect  it.  When  gently  warmed,  chrysophanic 
acid  reduces  the  ammonium  nitrate  of  silver.1 


FATTY  OILS 

Form  strongly  refractive  spherical  masses,  soluble 
in  ether,  carbon  bisulphide,  benzole  and  oil  of 
turpentine.  They  form  soap  with  potash  or  sodium 
lyes.  Perosmic  acid,  if  not  too  dilute,  colors  them 
black  or  brown. 

When  very  closely  united  with  protoplasm,  fats 
can  be  separated  by  concentrated  sulphuric  acid 
or  an  aqueous  solution  of  chloride  of  calcium, 
when  they  collect  in  drops  of  varying  size,  espe- 
cially at  the  margin  of  the  preparation. 

VOLATILE,   ETHEREAL,    OR  ESSENTIAL   OILS 

Form  ropy,  motile,  refractive  masses,  often  very 
long  in  proportion  to  their  diameter.  They  are 

l  Cf.  Schwarz:  Cohn's  Beitr.  z.  Biol.  d.  Pfl.,  1880,  III.,  p.  249. 


94  VEGETABLE    SUBSTANCES. 

soluble  in  cold  alcohol,  and  in  the  substances 
already  mentioned  as  solvents  of  fatty  oils ;  but 
they  are  insoluble  in  water. 


RESIN. 

The  best  marked  reaction  is  the  red  color  im- 
parted by  Miiller's  tincture  of  alcannin  (p.-4i). 
Hanstein's  aniline  violet  colors  it  blue  (p.  60). 
The  different  varieties  of  resin  are  soluble  in 
alcohol  and  ether,  but  insoluble  in  water.  The 
Unverdorben-Franchimont  reagent,  an  aqueous 
solution  of  acetate  of  copper  (p.  42),  colors  the 
resin  a  beautiful  emerald  green  in  masses  of  tissue 
which  are  allowed  to  lie  in  the  fluid  for  several 
days. 

WAX 

Forms  solid  crusts,  or  characteristically -shaped 
secretions,  on  the  surface  of  cells.  It  is  insoluble 
in  water,  whether  cold  or  hot,  but  melts  in  the 
latter  case.  It  is  insoluble,  or  hardly  soluble,  in 
boiling  alcohol,  but  soluble  in  ether,  benzole,  chlo- 
roform and  carbon  bisulphide. 


SILICIC  ACID    (Silica). 

Silica  frequently  incrusts  the  cell-wall  (grasses) 
or  the  pedicel  of  cystoliths,  and  it  is  sometimes 
found  in  the  interior  of  cells  in  amorphous  masses 


SILICA.  95 

(Podostemaceae).1  It  maybe  recognized  in  the  fol- 
lowing way :  A  very  thin  section  of  the  tissue  to  be 
tested  is  heated  on  platinum-foil,  by  which  means 
all  of  its  organic  constituents  are  destroyed,  leav- 
ing the  inorganic  elements,  which  consist  of  silicic 
acid  and  salts  of  lime,  in  a  somewhat  distorted 
skeleton.  The  calcareous  portion  is  then  removed 
by  adding  a  drop  of  hydrochloric  acid,  the  silica 
remaining  as  a  network,  which  is  soluble  in  hydro- 
fluoric acid. 

It  may  happen  that  the  silica  and  lime  salts  fuse 
together  in  the  process  of  heating,  which  inter- 
feres with  the  reaction.  It  is,  therefore,  desirable 
to  remove  these  salts  by  the  Schultze  maceration 
before  incineration.  When  the  section  has  been 
boiled  in  this  it  is  washed  in  hot  distilled  water. 
After  the  final  addition  of  hydrochloric  acid  the 
silica  alone  remains.  The  operation  needs  to  be 
skilfully  performed.  The  epidermis  of  Equisetum 
is  to  be  recommended  for  experiment. 

Sachs  has  proposed  a  somewhat  modified  method. 
Larger  tissue  masses  are  moistened  with  concen- 
trated sulphuric  acid  on  platinum-foil,  and  heated 
over  a  Bunsen  burner.  The  acid  is  at  once 
blackened,  while  there  is  a  violent  evolution  of  gas. 
The  heating  is  continued  only  until  the  pure  white 
ashes  remain,  —  a  result  which  is  reached  somewhat 
more  rapidly  by  this  method  than  by  the  other. 

1  Carlo:  Bot.  Zeitung,  1881,  p.  31.  Warming:  Naturh.  Forenings 
vidsk.  Meddel.,  Copenhagen,  1881,  p.  89. 


96          VEGETABLE  SUBSTANCES. 


LIME  SALTS 

Occur  partly  as  invisible,  amorphous  incrusting 
substances  in  the  cell-wall,  and  can  then  be  de- 
tected in  the  ashes.  Sometimes  they  occur  as 
well-developed  crystals,  which  are  found  in  the 
wall  itself  and  elsewhere.1 

Micro-chemical  investigations  show  that  calcium 
occurs  in  the  cell  in  the  form  of  carbonate,  oxalate, 
phosphate,  and  sulphate  :  — 

a.  The  compounds  with  carbonic  acid   are   dis- 
solved by  dilute  acids  with  violent  effervescence. 
If   the   solution   has   been   effected   by  sulphuric 
acid,  gypsum  needles  crystallize  out  in   the  fluid 
as  in  the  case  of  other  salts  of  lime. 

(Cystoliths ;  Corallina,  Melobesia,  Chara,  etc.) 

b.  Oxalate  of  lime,  the  most  widely-distributed 
of  these  compounds,  is  not  dissolved  by  potassic 
hydrate  or  acetic   acid,  but  it  is  soluble  in  hydro- 
chloric acid  without  effervescence. 

(It  occurs  as  an  incrusting  material  in  the  hairs 
of  Asclepias,2  and  in  raphides,  free  crystals, 
Rosanoff's  crystal  glands,  etc.) 

c.  Calcium  phosphate  3  has  recently  been  shown 
to  occur  in  plants.     It  is  insoluble  in  water,  alco- 

1  Holzner :   Beitr.   z.    Kenntn.    der    Pflanzenkrystalle.  —  Zeitsclir.    f. 
Mikroskopie,  1877,  !•>  P-  236,  where  there  is  a  synopsis  of  the  literature. 
—  Van  der  Ploeg:  De  oxalsure  Kalk  in  de  Planten,  Leiden,  1879. 

2  Kabsch  :  Bot.  Zeitung,  1863. 

3  Nobbe,  Hanlein,  and  Councler :  Landwirtsch.  Versuchsstationen,  Bd. 
XXIIL,  p.  471  (Robinia  Pseudacacia  and  Soya  hispida). 


IRON.  97 

hoi,  ether  and  alkalies,  but  soluble  without  effer- 
vescence in  acetic  and  other  acids.  The  character- 
istic reaction,  however,  is  the  yellow  color  pro- 
duced in  a  dilute  neutral  solution  of  nitrate  of 
silver. 

d.  Calcium  sulphate  (gypsum)  is  insoluble  or 
hardly  soluble  in  hydrochloric,  nitric  and  acetic 
acids.  When  crystals  of  this  salt  are  placed  in  an 
aqueous  solution  of  barium  chloride  they  are  soon 
covered  by  a  granular  crust  of  barium  sulphate.1 


IRON 

Has  been  detected  in  the  cell-wall.2  The  sections 
must  be  made  with  a  platinum  or  silver  knife. 
They  are  then  treated  with  an  alcoholic  tincture 
of  sulpho-cyanate  of  potassium  (p.  40),  and  the  ap- 
pearance of  a  red  color,  either  immediately  or  on 
the  further  addition  of  hydrochloric  acid,  indicates 
the  presence  of  a  ferric  compound.  In  case  no 
reaction  occurs  the  sections  are  treated  with  hydro- 

1  N.  J.  C.  Miiller  claims  to  have  found  this  salt  in  Guaiacum  wood. 
( Allg.  Bot.,  1880,  Theil  i,  p.  557.)     Hanbury  and  Fliickiger,  however,  only 
mention  calcium  oxalate  in  this  plant.     ( Pharmacographia,  1874,  P-  94-) 
Na'geli  (Mikroskop,  1877,  p.  486)  is  of  the  opinion  that  it  does  not  exist  in 
the  cell  at  all ;  while  Wiesner  (Techn.  Mikroskopie,  p.  85)  figures  the  twin- 
crystals  of  gypsum  from  the  mesophyll  of  Iris.     Holzner,  however,  in  his 
classic  work  on  the  crystals  of  plants,  shows  that  the  crystals  taken  for 
sulphate  of  lime  in  Guaiacum,  Iris,  etc.,  are  in  reality  the  oxalate.    The 
existence  of  crystals  of  the  sulphate  in  plants  is,  therefore,  not  demon- 
strated. 

2  Weiss  and  Wiesner:  Sitzungsber.  der  wiener  Akad.,  1860,  Bd.  XL. 
—  Cf.  Bot.  Zeitung,  1860,  p.  357. 


98  VEGETABLE    SUBSTANCES. 

chloric  or  nitric  acid,  together  with  the  sulpho- 
cyanate  (p-4o),  by  which  ferrous  compounds  which 
may  be  present  are  oxydized,  when  their  presence 
is  indicated  by  the  above  red  color. 


SULPHUR 

Has  been  found  in  the  pure  crystalline  state  only 
in  those  bacteria  which  live  in  thermal  springs  or 
on  rotting  algae,  and  which  liberate  sulphuretted 
hydrogen. 

Sulphur  is  soluble  in  carbon-bisulphide.  For 
the  violet  reaction  with  the  nitro-prussiate  of  so- 
dium in  alkaline  fluids  see  page  39.  It  gives  no 
reaction  in  an  aqueous  solution  of  perosmic  acid, 
and  thus  may  be  easily  distinguished  micro-chemi- 
cally  from  the  fatty  oils,  to  which  it  frequently 
bears  no  small  resemblance. 


COLORING    MATTERS.  99 


COLORING  MATTERS.1 


A.  —  Of  Protoplasm. 

CHLOROPHYLL  (Leaf  Green) 

Is  a  green  substance  which  is  insoluble  in  water, 
dilute  acids,  and  alkalies,  but  soluble  in  ether, 
benzole  and  alcohol.  It  is  bleached  by  Labar- 
raque's  solution.  When  treated  with  dilute  acids 
it  assumes  a  yellowish  color,  while  concentrated 
hydrochloric  and  sulphuric  acids  change  it  to  a 
bluish-green  or  blue.2 

Methyl  green  (p.  63)  colors  chlorophyll  bodies  a 
more  intense  green,  and  by  prolonged  treatment 
with  hydrochloric  acid  masses  of  hypochlorin  may 
be  extracted  from  them. 

According  to  Pringsheim,  the  following  pigments 
may  be  regarded  as  modifications  of  chlorophyll :  — 

a.     ETIOLIN, 

The  yellow  matter  of  etiolated  plants,  which  is 
soluble  in  alcohol  and  ether,  but  insoluble  in  water. 
Its  solution  is  colored  emerald  or  verdigris  green, 

1  Nageli :  Das  Mikroskop,  1877,  p.  528.  —  C/.,  further,  the  literature  of 
vegetable  coloring  matters,  srifra,  p.  xviii. 

2  Cf.  Bommer:  Bull,  de  la  Soc.  hot.  de  France,  1873,  Session  extraordi- 
naire. 


IOO         VEGETABLE  SUBSTANCES. 

and  later,  often  after  a  number  of  hours,  blue,  by 
hydrochloric  or  sulphuric  acid. 


b.     XANTHOPHYLL  (B'erzelius,  Pringsheim), 

The  yellow  coloring  matter  of  autumnal  leaves,  is 
insoluble  in  water,  but  soluble  in  alcohol  and  ether. 
It  is  only  colored  emerald  green  by  the  acids  which 
have  been  named. 


c.     ANTHOXANTHIN  (Marquart,  Pringsheim), 

The  yellow  pigment  of  yellow  flowers,  fruits  and 
seeds,  occurs,  like  chlorophyll,  diffused  through 
protoplasmic  bodies  ;  less  frequently  in  the  form  of 
yellow  oily  drops;  and  very  rarely  diffused  in  the 
cell-sap.  The  latter  variety  (anthochlor,  Prantl  ; 
xanthein,  Fremy)  is  soluble  in  water,  in  which  the 
other  forms  are  insoluble,  though  they  are  soluble 
in  ether  and  alcohol.  The  varieties  of  anthoxan- 
thin  (xanthein,  Fremy;  lutein,  Thudichum), which 
are  soluble  in  the  latter  reagents,  are  colored  green 
like  xanthophyll,  but  later,  like  etiolin,  blue,  by  the 
action  of  acids  ;  those  soluble  in  water,  however, 
are  not  affected  in  this  way.  Anthochlor  becomes 
brownish  yellow  when  treated  with  potassic  hy- 
drate, but  the  original  color  returns  when  the 
solution  is  neutralized. 


COLORING   MATTERS.  IOI 

d.     FLORIDEA-GREEN   (Pringsheim). 

The  chlorophyll  of  Florideae,  which  gives  the 
same  chemical  reactions  as  the  true  chlorophyll  of 
higher  plants,  is  to  be  regarded,  according  to 
Pringsheim,  as  a  variety  of  this,  on  account  of  its 
optical  properties. 

e.     FLORIDEA-RED   (Phycoerythrin,  Kiitzing,  Cohn) 

Is  soluble  in  water,  by  which  it  may  be  removed 
from  the  dead  plasma-bodies.  When  allowed  to 
stand  in  the  light,  exposed  to  the  air,  it  fades,  and 
the  same  effect  is  produced  when  it  is  treated  with 
potassic  hydrate.  Sulphuric  acid  does  not  change 
the  color.1 

HYPOCHLORIN.2 

After  chlorophyll  grains  have  been  treated  for 
some  hours  with  hydrochloric  acid  a  substance 
separates  from  them,  appearing  either  as  a  crystal- 

1  Nageli  and  Schwendener  (Mikroskop,  1867,  p.  498)  understand  by 
"  Floridea-Red  "  the  entire  coloring  mass  of  the  Florideae,  —  chlorophyll- 
phycoerythrin.     Sachsse  used  the  name  in  Kiitzing's  sense,  as  we  do.     Kiit- 
zing's  phycohaematin  from  Rhytiphloea  tinctoria,  which  needs  further  in- 
vestigation, is  omitted  here. 

NOTE.  —  To  distinguish  the  colors  which  have  been  mentioned,  properly, 
they  must  be  investigated  by  the  spectroscope.  Cf.  R.  Sachsse :  Farbstoffe, 
Kohlenhydrate  and  Proteinsubstanzen,  Leipzig,  1877. 

2  Pringsheim  :  Untersuchungen  iiber  Chlorophyll,  IV.,  Das  Hypochlorin 
und  die  Bedingungen   seiner  Entstehung  in  der  Pflanze.  —  Monatsber.  d. 
berl.  Akad.,  Nov.  1879.     (£/•  ibid.,  July  1879;  also  Comptes  rendus,  Jan. 
1880,  p.  161.);  Jahrb.  f.  wiss.  Bot.,  1882,  XIII.     A.  Hansen  :   Assimilation 
und  Chlorophyllf  unction,  1882. 


IO2  VEGETABLE    SUBSTANCES. 

line  deposit,  in  brown  oily  drops,  or  in  the  form  of 
ropy,  semi-fluid  masses,  from  which,  after  a  while, 
needle  or  staff-shaped  bodies,  or  fine  crumpled 
threads  separate.  According  to  Meyer '  the  hypo- 
thetical matter  called  hypochlorin  by  Pringsheim 
is  identical  with  the  chlorophyllane  of  Hoppe- 
Seyler,2  which  is  but  a  transformation  product  of 
the  green  pigment  of  the  living  plant,  and  perhaps 
also  identical  with  the  "crystallized  chlorophyll" 
of  Gautier. 

It  is  insoluble  in  water,  salt  solutions,  and  dilute 
organic  or  mineral  acids.  It  is  easily  soluble  in 
ether,  benzole,  volatile  oils  and  carbon  bisulphide. 
An  aqueous  solution  of  chloral  dissolves  the  crys- 
tals, leaving  a  drop  which  is  soluble  in  alcohol. 
Heat  volatilizes  it  so  that  green  cells,  which  have 
been  warmed  to  50°  C,  give  no  trace  of  hypo- 
chlorin when  subsequently  treated  with  hyrochloric 
acid.  When  hypochlorin  needles  that  have  been 
formed  by  the  aid  of  this  acid  are  heated  in  water 
they  lose  their  crystalline  nature,  and  unite  to 
form  oily  masses  of  a  greener  hue. 


CHLORORUFIN    (Rostafinski).* 

The    oospores   of    several    algae    (Oedogonium, 
Vaucheria),  the  antheridia  of  Chara,  and  the  cells 

1  A.  Meyer:  Das  Chlorophyllkorn,  Leipzig,  1883. 

2  Zeitschr.  f.  phys.  Chemie,  III. 

3  Rostaf.nski:  Bot.  Zeitung,  1881,  p.  461. 


COLORING    MATTERS.  IO3 

of  certain  fleshy  fruits  (e.  g.  Capsicum)  are  Col- 
ored red  by  this  substance.  Sulphuric  acid  gives 
it  a  very  intense  blue  color,1  quite  characteristic. 
Fuming  nitric  acid  dissolves  chlororufin,  while  ordi- 
nary nitric  acid  does  not.  This  reaction  indicates 
a  striking  analogy  with  the  chrysoquinone  of  Lie- 
bermann.  Chlororufin  is,  perhaps,  identical  with 
the  solanorubrin  of  Millardet,2  and  probably  is  a 
constituent  part  of  the  xanthein  of  Fremy. 


CAROTIN    (Wachenroder,   i832).3 

This  substance  forms  red  or  yellowish-red  crys- 
talline pigment  bodies,  without  any  organized  base, 
in  the  cells  of  Daucus  Carota.  It  has  also  been 
obtained  in  the  free  state.  It  is  easily  soluble  in 
benzole,  carbon  bisulphide  and  fatty  arid  volatile 
oils.  Ether  and  alcohol  dissolve  it  with  difficulty. 
Chloroform  dissolves  it  readily;  but  an  aqueous 
solution  of  chloral  (5  : 2)  or  acetic  acid  does  not 
produce  any  alteration.  Sulphuric  acid  produces 
a  distinct  blue  color,  and  chloride  of  iron  gives 
a  greenish  color  with  a  solution  of  this  sub- 
stance. 


1  De  Bary:  Ber.  d.  natiirf.  Ges.,  Freiburg,  1856,  No.  13. 

2  Millardet:  Note  sur  une  substance  colorante  nouvelle,  Nancy,  1876. 

3  A.  Husemann :  Ueber  Carotin  und  Hydrocarotin,  Diss.,   Gdttingen, 
1860.     A.    Meyer:    Das  Chlorophyllkorn,  1883,  p.  48.      Fritsch :  Ueber 
kornige  Stoffe  des  Zellinhaltes,  Diss.,  Konigsberg,   1882,  p.  39.     Zeise : 
Jahrb.   f.  Chemie,  XL.,  p.  297.     Berg:    Pharmaceutische  Waarenkunde, 
1879. 


IO4  VEGETABLE    SUBSTANCES. 


PHYCOCHROMIN  (Sachsse  =  Na'geli's  phycochrome  — 
chlorophyll) 

Is  a  pigment  which  occurs  in  connection  with 
chlorophyll  in  the  bluish-green  algae.  It  is  soluble 
in  water,  but  insoluble  in  alcohol,  and  probably 
consists  of  .  phycocyanin  (Sachsse x),  and  a  variable 
quantity  of  phycoerythrin  (Kiitzing).  Cells  which 
contain  phycochrome  are  colored  yellowish-green 
or  yellowish  -  brown  by  alkalies,  and  orange  or 
brick-red  by  hydrochloric  acid. 


PALMELLIN  (Phipson2) 

Is  the  red  pigment  found  in  Porphyridium  cruen- 
tum  Naeg.  It  is  soluble  in  water.  The  addition 
of  alcohol,  acetic  acid  or  alkalies  to  this  solution 
causes  a  flocculent  precipitate,  while  the  fluid 
assumes  a  blue  color.  Ammonium  sulphide  colors 
it  yellow  without  forming  a  precipitate. 

PHYCOXANTHIN   (Millardet,  Askenasy) 
Is   the   yellow   coloring    matter   of    diatoms   and 
Fucaceae,  and  is  more  readily  soluble  in  alcohol 
than  chlorophyll  is.     In  the  diatoms  it  forms,  with 
chlorophyll,    the      yellowish-brown      endochrome 

1  Kiitzing's  phycocyanin  is  the  coloring  matter  of  Oscillarise,  which  is 
soluble  in  water. 

2  Comptes  rendus,  Aug.   1879,  p.   316.    Schnetzler:  Bull,  de  la  Soc. 
vaudoise  des  Sc.  nat..  2.  Ser.,  Vol.  XV. 


COLORING   MATTERS.  IO5 

masses,1  and  has  been  called  diatomin  (Nageli, 
1849).  ft  is  easily  removed  from  the  thallus  of  the 
Fucaceae  by  40  per  cent,  alcohol,  which  does  not 
extract  the  chlorophyll.  A  small  quantity  of  acid 
colors  it  bluish-green.  Alkalies,  like  light,2  have 
no  pronounced  influence  on  it. 


PHYCOPHAEIN  (Millardet) 

Occurs  in  the  thallus  of  Fucaceae,  mixed  with 
chlorophyll  and  phycoxanthin.  It  is  brown,  soluble 
in  water  but  insoluble  in  alcohol.  Further  inves- 
tigations, however,  are  necessary. 


B.—  Of  the  Cell  Sap. 

(ANTHOXANTHIN.  —  For    a    form    of    this,    see 
page  100). 

ANTHOCYANIN  (Marquart;  Kyanin,  Fremy  and  Cloez) 

Is  the  blue  coloring  matter  of  many  flowers.  The 
erythrophyll,  which  is  peculiar  to  the  cell-sap  of 
red  and  violet  cells,  is  probably  identical  with  this, 
or  only  a  modification  of  it.  Acids  impart  a  red 
color  to  cells  which  contain  anthocyanin,  while 
alkalies  restore  the  blue  color,  the  same  as  with 

1  Petit:  De  1'Endochrome  des  Diatome'es.  —  Bre"bissonia,  1880,  Ann. 
II.,  No.  7,  p.  81.  —  The  composition  of  the  endochrome  was  established  in 
1868  by  Kraus  and  Millardet. 

2  Cf.,  however,  Petit,/,  c. 


IO6  VEGETABLE    SUBSTANCES. 

litmus.  After  the  prolonged  action  of  alkalies 
the  blue  passes  into  a  green,  yellowish-green,  or 
yellow.  But  it  has  been  thought  that  the  latter 
tints  are  produced  in  consequence  of  the  presence 
of  tannin  in  the  cell-sap,  which  gives  a  green  color 
with  iron,  and  which  may  give  the  yellow  potassic 
hydrate  reaction  in  the  color  mixture.1 

In  Strelitzia  Reginoe  and  Tillandsia  amoena, 
Hildebrand  found  anthocyanin  diffused  in  small 
grains,  which  were  soluble  in  potash,  alcohol,  and 
ammonia  ;  which  iodine  colored  brown  ;  and  which 
were,  therefore,  probably  of  a  protoplasmic  nature. 

It  is  doubtless  worthy  of  mention  here  that 
anthocyanin  may  form,  with  metallic  salts,  an 
insoluble  compound  of  a  green  (Fremy  and  Cloez) 
or  blue  (Wiesner)  color.  Certain  cells  (Gentiana 
verna)  containing  anthocyanin,  which  have  first 
been  reddened  by  acids  that  have  then  been  re- 
moved by  rinsing  with  distilled  water,  assume  a 
blue  color  with  a  solution  of  chloride  of  iron. 
Since,  however,  the  same  phenomenon  occurs  with 
acetate  of  lead,  tannic  acid  can  have  nothing  to 
do  with  it. 

ALIZARIN  2 

Forms  yellow  masses  in  the  fresh  cells  of  madder- 
root.  With  the  access  of  air  it  soon  becomes  red 

1  Sachsse's  objections,  however,  should  be  compared  with  this,  /.  c .,  p.  76. 

2  Decaisne:    Recherches  anat.  et  physiol.  sur  la  Garance  et  le  deve- 
loppement  de  sa  matiere  colorante,  10  pi.,  Bruxelles,  1837.     Rosenstiehl: 
Ann.  de  Chim.  et  de  Phys.,  5  Ser.,  T.  XIII.,  p.  148. 


COLORING   MATTERS.  IO/ 

and  flocculent  and  passes  into  the  cell -walls. 
Potassic  hydrate  colors  it  purple,  and  causes  its 
escape  into  the  surrounding  medium.  Chloride 
of  iron  colors  it  orange  and  finally  brownish-red. 
Alcohol  dissolves  the  fresh  yellow  pigment,  but 
not  that  which  has  been  reddened  by  exposure  to 
the  air. 

INDICANE,  INDIGOTIN. 

Although  the  chemical  investigation  of  this 
substance  in  the  living  cell  has  so  far  been  very 
unsatisfactory,  it  will,  no  doubt,  be  of  inter- 
est to  histologists  for  us  to  call  attention  to  it 
again. 

In  the  cells  of  the  flowers  of  certain  orchids, 
especially  Phajus,  a  blue  substance  is  formed  when 
the  plant  dies.  It  occurs  either  as  distinct  crystals 
or  groups  of  crystals,  and  as  small  but  numerous 
granules.  These  may  be  readily  produced  by  crush- 
ing the  parts  of  the  flower,  or  by  treating  them 
with  alcohol.  Micro-chemical  analysis  shows  this 
substance  to  be  indigotin ;  it  is,  therefore,  proba- 
ble that  indicane  previously  existed  in  the  cells, 
possibly  in  small  protoplasmic  (?)  granules  (Tro- 
phoplasts  ?).  The  only  micro-chemical  reaction 
for  this  substance  at  present  known  is  its  forma- 
tion by  alcohol.  It  can  be  sublimated,  and  after- 
ward forms  small  crystals.1 

1  Cf.  Bommer :  Bleuissement  des  fleurs  de  Phajus,  etc. ;  Just's  bot. 
Jahresbericht,  1874,  II.,  2,  p.  868. 


IO8         VEGETABLE  SUBSTANCES. 


C.  —  Of  the   Wall. 

THE  PIGMENTS  OF  DYE-WOODS  (Brazil  Wood, 
Sandal-Wood,  etc.) 

Occur  in  the  cell-contents  as  well  as  in  the  wall, 
especially  in  the  middle  lamella,  which  may  not 
improbably  have  derived  them  from  the  former. 
They  are  soluble  in  warm  water,  glycerine,  acids 
and  alkalies.  "  Alkalies  dissolve  the  pigments 
very  easily,  with  the  production  of  a  carmine  or 
violet  color.  Alcohol  and  ether  give  either  a 
colorless  (Brazil  wood,  etc.)  or  a  yellow,  orange, 
or  carmine  solution.  Glycerine  and  water  dis- 
solve them  with  a  carmine,  or,  less  frequently, 
with  a  blood-red,  brownish-red,  or  violet  color ; 
cuprammonia,  with  a  violet,  or,  more  rarely,  with  a 
blue  color ;  and  acetic  acid  with  a  yellow  color. 
Sulphuric  acid  either  does  not  dissolve  them  at 
all  (sandal-wood),  or  with  the  production  of  a  car- 
mine or  blood-red  color."  x'  The  coloring  matter 
of  species  of  Pterocarpus  is  exceptionally  insoluble 
in  hot  water. 

THE  COLORING  MATTER  OF  THE  BARBERRY 
ROOT 

Is  yellow,  and  occurs  in  the  walls  of  the  ducts, 
wood-cells,  medullary  rays,  bast-cells  and  the  ex- 
ternal cortical  cells.  It  is  also  found  in  the  cell- 

1  Weiss. :  Allg.  Bot,  L,  p.  137. 


COLORING   MATTERS.  ICX) 

contents  of  all  the  tissue  systems,  with  the 
exception  of  the  spiral  ducts. 

Dilute  acids  induce  at  first  the  separation  of 
small  yellow  drops,  which  exhibit  the  Brownian 
movement.  Hot  potash  produces  a  brownish- 
yellow  color.  Glycerine  and  water  dissolve  it. 
Dried  sections  are  rapidly  extracted  by  water. 
In  this  yellow  fluid  hydrochloric  acid  forms  yel- 
low, often  radiate  aggregates  of  chloride  of  ber- 
beridin. 

GLOCOCAPSIN 

Is  a  red  or  blue  pigment  occurring  in  the  cell- 
walls  of  Gloeocapsa  and  certain  filamentous  algae. 
It  changes  to  a  red,  or  brownish-red,  under  the 
action  of  hydrochloric  acid,  and  to  blue  or  violet 
when  acted  upon  by  potash. 


SCYTONEMIN 

Is  a  yellow  or  brown  coloring  matter  which  occurs 
in  the  cell-walls  of.  many  Phycochromaceae.  It  is 
changed  to  a  verdigris-green  by  hydrochloric  acid, 
the  yellow  color  reappearing  on  the  addition  of 
alkalies. 


INDEX 


INDEX. 


A. 

Acetic  acid,  n,  12,  22,  48,  54,  cc, 
63,  66,  83,  85,  89,  92,  96, 
97,  104,  1 08. 

Acetic  acid  glue,  72. 

Achromatin,  57. 

Air,  removal  of,  from  sections,  25. 

Albumen  of  seeds,  12. 

Albuminoids.  See  protein  sub- 
stances. 

Alcanna  tinctoria,  48. 

Alcannin,  48,  94. 

Alcohol,  10,  25,  34,  45,  49,  50,  51, 
52>  63,  71,  76,  78,  79,  82, 
83,  85,  88,  91,  94,  99,  100, 
103, 105, 106,  107,  1 08. 

Aldehyde,  81. 

Aleuron  grains.  See  protein 
grains. 

Algae,  mounting,  67-9. 
preparing  for  study,  14. 

Alizarin,  106. 

Althaea,  45. 

Alum,  12,  37,  48,  53,  56. 

Alum  carmine,  50,  76,  77,  78,  83. 

Ammonia,  n,  13,  14,  15,  17,  40, 

47,  49,  5°,  52>  54,  55,  78, 
80,  82,  89,  92. 
Ammonium,  carbonate,  93. 
carminate,  49,  53,  83. 
cupro-sulphate.     See   ciipram- 

monia. 

hydrate.     See  Ammonia. 
picro-carminate,  53. 
sulphide,  104. 
Amyloids,  60. 
Amyloid,  5,  16,  76,  90. 
Amylomycin,  85. 
Anhydrating  tissues,  27, 37,  44,  70. 


Anilin,  63,  90. 

chloride,  43. 

sulphate,  42,  77. 
Aniline  colors,  58,  76,  78. 

blue,  61. 

brown,  62. 

green,  63,  83. 

violet,  59,  82,  89,  90,  94. 
Anthochlor,  100. 
Anthocyanin,  105 
Anthoxanthin,  100,  105. 
Aqua-fortis.     See  nitric  acid. 
Arabin,  37. 
Arsenious  acid,  65. 
Asclepias,  96. 
Asparagin,  26,  45,  91. 
Asphalt,  67,  71. 
Autumn  leaves,  color,  100. 


B. 

Bacillus,  57,  59,  62,  63. 
Bacteria,  killing,  7. 

staining  and  mounting,  24,  56, 
58,  60,  62. 

sulphur  in,  98. 
Bacterium,  57. 
Balsam  of    Firs.      See  Canada 

balsam. 

Barberry  pigment,  108. 
Barium,  chloride,  97. 

hydrate,  93. 
Bassorin,  37. 
Bast,  48,  60,  108. 
Benzole,  29,  34,  70,  72,  78,  92>  93, 
94,  99,  102,  103. 
Benzole  balsam,  67, 69. 
Berberidin  chloride,  109. 
Berberis,  108. 


INDEX. 


Bertholletia,  32. 
Borax,  52. 
Brazil  wood,  108. 
Brucin,  46. 

Brunswick  black,  67,  71. 
Butter,  cocoa,  20. 

C. 

Calcium,  carbonate,  22,  96. 

chloride,  32,  68,  85,  93. 

hydrate,  93. 

oxalate,  96. 

phosphate,  96. 

sulphate,  97. 
Callous  plates,  47,  61. 
Campeachy  wood,  56. 
Camphor,  67. 

Canada  balsam,  20,  56,  57,  61,  62, 
64,  67,  69,  71. 

Cane  sugar.     o<<?£  saccharose. 
Caoutchouc,  92. 
Capsicum,  103. 

Carbolic  acid,  24,  53,  67,  77,  85. 
Carbon  bisulphide,  29,  92-94,  98, 
102,  103. 

Carbonates,  22,  96. 
Carmine,  49. 

Carmine     preparations,     mount- 
ing, 23. 

Carmine,  picro,  53. 
Carminate  of  ammonium,  49,  ^3, 

83- 

Carotin,  103. 

Cells  for  mounting  objects,  66. 
Cell-sap,  105. 
Cell-wall,  1 8,  108. 
removal  of  incrusting  substan- 
ces, 6,  13,  77. 

reagents  which  cause  swelling, 
5,  21,  76. 

stratification,  12,  19. 
striation,  12,  38. 
middle  lamella,  12,  14,  15,  17, 
18,  21,  34,  77,  91,  108. 
Cellulose,  8,  15   16,  37,  50,  51,  60, 

61,  75,  77- 

Cellulose,  fungus,  9,  79. 
Cellulose,  starch,  4,  84. 
Cerasin,  37. 
Ceric  acid,  18,  34,  78. 
Chara,  21,  96,  102. 
Cherrywood  extract,  47,  77. 


Chloral,  43,  55,102. 
Chloroform,   2g,   31,   34,  78,   92, 
94,  103. 

Chloroform  balsam,  69. 
Chloro-iodide  of  zinc,  8,  75,  76, 
78,  90. 

Chlorophyll,  99. 
Chlorophyll,  crystallized,  102. 
Chlorophyll  bodies,  7,  22,  63. 

starch  in,  7. 
Chlorophyllane,  102. 
Chloro  rufin,  102. 
Chromatin,  57. 

Chromic  acid,  18,  20,  56,  76-78, 85. 
Chromoplasts,  7,  22,  63. 
Chrysoidin,  63. 
Chrysophanic  acid,  13,  93. 
Chrysoquinone,  103. 
Cilia,  7. 
Clearing  tissues,  n. 

for  study,  it,  12,  27,  31,  32,  37, 

43- 

for  mounting,  20,  27,  52,  70. 
Clove  oil,  20,  31. 
Cochineal,  23,  48,  53. 
Cocoa  butter,  20. 
Collenchyma,  12,  16. 
Colleters,  60. 
Coloring  agents,  48. 
Coloring  matters  of  plants,  99. 
Coniferin,  76. 

Consumption  Bacillus,  59,  62,  63. 
Copper,  15. 

acetate,  42,  87,  94. 

hydrate,  15. 

sulphate,  13,  15,35,  76. 
Coprinus  evanidus,  86. 
Coralline,  96. 
Cork,  6,  9,  12,  19,  78. 
Corrosive  sublimate,  32,  80. 
Cotton,  15. 
Crenothrix,  39. 
Creosote,  90. 
Crystals,  22,  23,  96. 
Crystalloids,  7,  13,  14,  21,  32,  91. 

in  protein  grains,  29. 
Culture  fluids,  37. 
Cuoxam.     See  cupr ammonia. 
Cuprammonia,  14,  76-78,  85,  88, 
91,  108. 

Cupridiamin.    See  ctiprammonia. 
Cuticle,  9,  15,  17,60. 
Cystoliths,  94,  96. 


INDEX. 


D. 

Dammar,  58,  72. 
Daucus  carota,  103. 
Desmids,  68. 
Dextrin,  17,  36,  87. 
Dextrose,  87. 
Diastase,  4. 

Diatoms,  6,  19,  22,  65,  69,  104. 
Diatomin,  105. 
Diphenylamin,  45. 
Double  staining,  51,  58,  59. 
Ducts,  6,  9,  1 08. 
Dye-wood  pigments,  108. 


Ectoplasm,  23,  25. 
Embryo,  n,  28. 
Embryo  sac,  44,  52,  82. 
Eosin,  57,  77. 
Epidermis,  9. 
Epiplasin,  82. 
Equisetum,  95. 
Erythrophyll,  105. 
Essential  oils.     See  oils. 
Ether,  29,  31,  34,-  7'i  7&,  93>  ( 
99,  TOO,  102,  103,  ic 
Ether  balsam,  69. 
Ethereal  oils.     See  oils. 
Ethyl  alcohol,  25.     • 
Etiolin,  99. 

Euphorbiaceae,  starch  of,  85. 
Exospore,  9,  17. 
Extine,  9. 


F. 

Farrant's  solution,  65. 

Fats  and  fatty  oils.    See  oils. 

Fehling's  test,  87. 

Ferric  salts.     See  iron. 

Fibro-vascular  bundles,  n,  27,  60. 

Fish  glue,  72. 

Florideae,  65,  92. 

Floridea  green,  101. 

Floridea  red,  101. 

Flowers,  blue,  105. 

Flowers,  yellow,  100. 

Fruits,  yellow,  100. 

Fruits,  red,  103. 

Fucacese,  104,  105. 

Fuchsin,  59,  78. 


Fungus-cellulose.  9,  70. 
Fungi,  38,  66.     ' 
Fungine,  79. 

G. 

Gelatine,  67. 

Gentiana  verna,  106. 

Glands,  nectar,  26. 

Glands,  resin,  60. 

Globoids,  29. 

Gloeocapsa,  109. 

Gloeocapsin,  109, 

Glucose,  36,  41,  87. 

Glue,  72. 

Glycerine,  3,  12,  16,  20,  23,  37, 

34,  36>  39,  49,  52,  56,  57, 
6 1,  62,  65,  67,  68,  82,  88, 
92,  1 08,  109. 

Glycerine  and  acetic  acid,  23. 

Glycerine  jelly,  52,  57,  67. 

Glycogen, 83, 

Gold  chloric 

Gold  size,  71. 

Gram-Riitzou  cement,  72. 

Granulose,  4,  84. 

Grape  sugar.     See  glucose. 

Grasses,  94. 

Guaiacum  wood,  97. 

Gums,  60,  65,  76,  89. 

Gypsum,  97. 

H. 

Haematoxylin,  55,  83. 

Hardening  resins,  40. 

Hardening  tissues,  25,  50,  68. 

Hesperidin,  26,  88. 

Hydriodic  acid,  5,  7. 

Hydrochloric  acid,  4,  8,  n,  21,  39, 
43.  47,  49,  68,  75,  76,  93, 
95-97,  99,  Joo,  i°4,  109- 

Hydrofluoric  acid,  95. 

Hypochlorin,  22,  99,  101. 


Incrusting  substances,  6,  13,  77. 

Indicane,  107. 

Indigotin,  107. 

Indol,  46,  77- 

Intercellular    substance,    12,    14, 

15,  17,  18,  21,  34,  77,  91, 

108. 


u6 


INDEX. 


Inulin,  7,  26,  28,  88. 
Iodine,  3,  8,  28,  75,  80,  83,  84,  88, 
90,  106. 

Iodine  green,  51. 
Iris,  97. 
Iron,  40,  97. 

acetate,  33,  42,  90. 

chloride,  33,  90,  103,  106,  107. 

ferric  salts,  39. 

sulphate,  33. 
Ivory  black,  72. 


Japan  varnish,  72. 


Kyanin,  105. 


K. 


L. 


Labarraque's  solution,  51,  99. 
Latex,  starch  in,  85. 
Laticiferous  tissue,  n. 
Lead  acetate,  106. 
Leaves,  clearing,  n. 

autumnal,  100. 
Lemon  oil,  31. 
Levulose,  88. 
Lichens,  4,  6,  So,  93. 
Lichenin,  80. 

Lignin,  6,  15,  19,  24,  42,  46,  47, 
50,  60,  76. 
Lime  salts,  96. 
Linseed  oil,  71. 
Logwood  extract,  55. 
Lupinus,  45. 
Lutein,  100. 

M. 

Maceration,  15,  17,  34,  76-78,  95- 

Madder  pigment,  106. 

Magenta,  63. 

Medullary  rays,  108. 

Melobesia,  96. 

Mercury,  38. 

.  chloride,  32,  80. 

nitrate,  38,  80,  86 
Meristem,  u,  20. 
Metacellulose,  79. 
Metaplasm,  82. 
Methyl  blue,  62. 


Methyl  green,  23,  63,  99- 
Methyl  violet,  59,  60,  69. 
Middle  lamella.     See  cell-wall. 
Millon's  reagent,  38,  80,  86. 
Monobromo-Naphthalin,  69. 
Monotropa,  44. 
Mordants,  37,  56. 
Mosses,  14,  24. 
Mucus,  89. 
Muriatic  acid,  21. 


N. 

Naphthalidin,  77. 
Nectaries,  26. 
Nicholson's  blue,  58. 
Nigrosin,  57. 
Niter,  37. 

Nitric  acid,  5,  13,  J7,  36, 
77,  78, 

Nitric  acid,  fuming,  103. 
Nitric  acid  glue,  72. 
Nitrates,  45,  46. 
Nitrites,  45,  46. 
Nucleus,  7,  22,  23,  49-54, 
60,  61,  63; 

division,  25,  38,  63. 

nuclear -plates,  63. 

achromatin,  57. 

chromatin,  57. 

nuclein,  83. 


O. 

Oedogonium,  102. 
Oils,  essential,  26,  30,  31,  43,  92, 
93,  102,  103. 

spirits  of  turpentine,  30,  71,  93. 

oil  of  cloves,  20,  31. 

oil  of  lemons,  31. 
Oils,  fatty,  n,  17,  20,  26,  30,  43, 
93,  98,  103. 

linseed  oil,  71. 
Oranges,  for  hesperidin,  89. 
Orchids,  107. 
Orchis,  44. 
Oscillaria,  104. 
Osmiamid,  20. 

Osmic  acid.     See  perosmic  acid. 
Ovule,  44. 

Oxalic  acid,  23,  50,  51,  91. 
Oxalates,  22,  96. 


56,  57, 
,  82,  83. 


INDEX. 


117 


P. 

Paeonia,  75. 

Palmellin,  104. 

Paste,  84. 

Pectin,  15,  24,  gi. 

Pepsin,  4. 

Perosmic  acid,  19,  68,  82,  83,  86, 

93>  98- 

Peziza  vesiculosa,  87. 
Phajus,  107. 

Phenol,  24,  53,  67,  77.  85. 
Phloem,  48. 
Phloroglucin,  46,  77- 
Phosphorus,  69. 
Phosphoric  acid,  5,  21. 
Photographing  bacteria,  61,  62. 
Phycochromaceae,  104, 109. 
Phycochrome,  104. 
Phycochromin,  104. 
Phycocyanin,  104. 
Phycoerythrin,  101,  104. 
Phycohaematin,  101. 
Phycomyces  nitens,  86. 
Phycophaein,  105. 
Phycoxanthin,  104. 
Picric  acid,  53-55. 
Picrocarmine,  53,  83. 
Pigments,  99. 
Podostemacese,  95. 
Pollen  grains,  14,  31,  68. 

extine,  9,  17. 
Pollen-cultures,  44. 
Porphyridium  cruentum,  104. 
Potash.     See  potassium  hydrate. 
Potassium,  acetate,  61,  69. 

arabate,  37. 

bichromate,  40,  68,  90. 

chlorate,  17,34- 

ferro-cyanide,  39,  83. 

hydrate,  5,  9,  21,  34,  39,  40,  49, 
76,  78,  79,  82,  85,  89,  92, 
93,  101,  106,  107,  109. 

iodide,  3,  5,  8,  75,  85. 

metagummate,  37. 

nitrate,  37. 

sulpho-cyanate,  40,  97. 

tartrate,  36. 
Prosenchyma,  48. 
Protein  grains,  7,  28,  33,  80,  92. 

substances,  14,  21,  37,  38, 

43>  44,  49-5 T>  79,  80. 

Protoplasm,  6,  u,  13,  17,  20,  23, 

3I»5J»54?58>6o>6l»8i,  99- 


Protoplasm,  living,  8r. 

fixation  of,  7,  19,  20,  23,  25. 

streaming,  32. 
Prussiate  of  potash,  39,  83. 
Pterocarpus,  108. 
Punctum  vegetationis,  n. 

R. 

Raphides,  96. 

Resins,  26,  30,  42,  60,  94. 

hardening,  40. 
Rhodospermin,  92. 
Rhytiphloea  tinctoria,  101. 
Robinia  pseudacacia,  96. 
Root  cap,  6,  9,  1 7. 
Rosanilin  sulphate,  58. 
Rosolic  acid,  47. 


Saccharose,  17,  26,  28,  35,  44,  80, 
82,  88. 

Salt,  table.     See  sodium  chloride. 
Saltpeter,  37.  _ 
Sandalwood  pigment,  108. 
Sarcina,  57. 
Sarcinoglobulus,  57. 
Schultze  maceration,  15,  17,  34, 
76-78,  95. 
Scytonemm,  109. 
Sealing  wax,  71. 
Seeds,  yellow,  100. 

albumen,  12. 

mucilaginous  coats,  5. 
Shellac,  7:,  72. 
Sieve  tubes,  47,  58,  60,  61. 
Silica,  6,  19,  94. 
Silicic  acid.    See  silica. 
Silver  nitrate,  40, 81,  87, 91,  93, 97., 
Sinistrin,  88. 
Sodium  lyes,  93. 

carbonate,  47. 

chloride,  31,  82,  92. 

hydrate,  15. 

nitro-prussiate,  39. 
Solanorubrin,  103. 
Soya  hispida,  96. 
Sphaeria  Desmazierei,  85. 
Sphaero-crystals,  7,  26,  28,  88. 
Spirogyra,  41.    . 
Spores,  preparing  for  study,  14. 

mounting,  68. 

exospore,  9,  17. 


iiS 


INDEX. 


Staining  agents,  48. 
Staining,  double,  51,  58,  59. 
Starch,  3,  4,7,9,11,12,  16-18,21, 
43,51,68,84. 

conversion  into  paste,  84,  85. 
Starch  grains, 

granulose,  4,  84. 

starch  cellulose,  4,  84. 

stratification,  19,  85. 
Stem,  clearing,  u. 
Stratification 

of  cell-wall,  12,  19. 

of  starch  grains,  19,  85. 
Streaming  of  protoplasm,  32. 
Strelitzia  Reginae,  106. 
Striation  of  cell-wall,  12,  38. 
Suberin,  6,  12,  15,  18,  19,  34,  50, 
78. 

Sublimate,  corrosive,  32,  80. 
Sugar,  cane.     See  saccharose. 
Sugar,  grape.     See  glucose. 
Sulphur,  39,  98. 
Sulphuretted  hydrogen,  98. 
Sulphuric  acid,  4,  5,  16,  21,  36, 
43~46,  49,   75-77,   8°,  90, 
93,  99-101,  103,  108. 
Syrup,  44. 
Synantherin,  88. 

T. 

Table  salt.     See  sodium  chloride. 
Tannic  acid.     See  tannin. 
Tannin,  9,  13,  20,  33,  60,  go,  106. 
Tin  chloride,  90. 
Thomsen's  wood-gum,  76. 
Thymol,  55,  63. 
Tillandsia  amcena,  ic6. 
Tissues, 
anhydrating,  20,  25,  56,  57,  64, 

i      •  7°' 

clearing,  n,  12,  20,  27,  31,  32, 

37,  43,  52,  7°- 
hardening,  25,  50,  68. 
Toluidin,  77. 
Trichomes,  clearing,  u. 


Trichlor-acetic  acid,  85. 
Trommer's  reagent,  35,  86-88. 
Trophoplasts,  107. 
Tuberculosis  Bacillus,  59,  62,  63. 
Turpentine,  30,  71   93. 
Tyrosin,  26. 

U. 
Urginea  scilla,  88. 

V. 

Vanillin,  76-77, 
Vaucheria,  102. 
Verdigris,  42. 
Vessels,  6,  9,  108. 
Vesuvin,  62. 
Vitriol,  blue,  35. 
Vitriol,  oil  of,  16. 
Volatile  oils.    See  oils. 


W. 

Wax,  26,  30,  94. 

Wax  cells,  66 
Wax,  sealing,  71. 
Wood,  42,  78. 

cells,  6,  9,  21,  34, 108. 

gum,  76. 

X. 

Xanthein,  100,  103. 
Xanthophyll,  100. 
Xanthoprotein    reaction,   13,   14^ 
17,  80. 

Y. 

Yeast,  68. 

Z. 

Zinc,  8. 
chloride,  5,  85. 
chloriodide,  8,  75,  76,  78,  90. 
iodide,  5. 


